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Identification of Protein Kinase C Isoforms Involved in Type 1 Diabetic Encephalopathy in Mice

Diabetic encephalopathy is a complication of diabetes mellitus characterized by impaired cognitive functions. Protein kinase C (PKC) isoforms are rarely reported on diabetic encephalopathy, although they have been believed to play crucial roles in other diabetic complications. In this study, streptozotocin- (STZ-) induced diabetic mice were found to exhibit learning and memory deficits in the Morris water maze test. Meanwhile, the expression of cPKCβII, nPKCε, and cPKCγ did not change in the hippocampus, cortex, and striatum at 2 and 8 weeks after STZ injection. The nPKCε translocation to the membrane, where it is activated, was not altered in the above brain regions at 2 and 8 weeks after STZ injection. Nevertheless, cPKCβII translocation to the membrane was significantly decreased in the cortex and hippocampus at 8 weeks after STZ injection. The translocation of cPKCγ from the cytosol to the membrane was remarkably decreased in the hippocampus at 2 and 8 weeks and in the cortex and striatum at 8 weeks after STZ injection. In addition, deletion of cPKCγ aggravated the impairment of spatial learning and memory. In conclusion, our results suggest that the decrease in the activity of cPKCβII and cPKCγ, especially cPKCγ, may play key roles in the pathogenesis of diabetic encephalopathy.

Introduction
The prevalence rates of diabetes mellitus (DM) have increased rapidly over the past three decades in the world [1]. Some clinical studies have shown that high blood glucose increases the risk of Alzheimer's disease (AD) [2][3][4]. It has been manifested that type 1 diabetes mellitus (T1DM) has shown neurofibrillary and senile plaque, two major pathologic characteristics of AD, in the animal models [5,6]. To describe cognitive impairment in diabetes, the term "diabetic encephalopathy" was introduced [7]. Now, diabetic encephalopathy is accepted as one of the most widespread diabetic complications in the central nervous system [8]. Because of a lack of effective treatment for diabetic encephalopathy, much interest now is focused on its molecular mechanism.
Constant hyperglycemia inside the cell increases the synthesis of diacylglycerol, which is one of the critical activating factors for protein kinase C (PKC) [9]. PKC, a family of Ser/Thr kinases that regulate a series of cellular processes, may play a key role in diabetes and its complications. According to the forms of activation, PKC isoforms are included in three groups: (1) conventional PKC isoforms (cPKCα, βI, βII, and γ) are activated by diacylglycerol, phosphatidylserine, and Ca 2+ influx; (2) novel PKC isoforms (nPKCδ, ε, η, and θ) require diacylglycerol; and (3) atypical PKC isoforms (aPKCζ and ι/λ) do not bind either second messengers. It has been demonstrated that the cPKCβII, cPKCγ, and nPKCε, widely expressed in the nervous system, participate in many diabetic complications [10][11][12][13][14].
cPKCβII is the most studied isoform for diabetic complications. Deletion or inhibition of the cPKCβII can reduce glomerular, albuminuria, and mesangial expansion, which prevent the thickening of the glomerular basement membrane and the obliteration of glomerular capillaries in a diabetic kidney [15,16]. The cPKCβII inhibitor can reverse acute blood glucose fluctuation-induced endothelial cell apoptosis increase, inflammatory cytokine level increase, and insulin signaling impairment, indicating that PKCβII may serve as a target for anticardiovascular diseases [11]. The cPKCβII inhibitor can also reduce the incidence of visual loss and prevent blood-retinal barrier breakdown in diabetic patients and animal models [17].
nPKCε is highly relative to insulin resistance. The key role of nPKCε for fat-induced hepatic insulin resistance is demonstrated with antisense oligonucleotide-mediated knockdown of nPKCε in high-fat feeding rats [18]. Several studies in type 2 diabetic patients have demonstrated that activation of nPKCε by hepatic accumulation of the diacylglycerol can result in hepatic insulin resistance and nonalcoholic fatty liver disease [19]. nPKCε deletion also enhances the amplifying pathways of glucose-stimulated insulin secretion and lipolysis in diabetic mouse beta-cells [20].
Compared to other PKC isoforms, cPKCγ is located only in the neurons of the central nervous system. Some studies have reported that the inhibitors of cPKCγ could reverse the DM-associated chronic impairment of neurovascular coupling [10]. The increase in cPKCγ expression in a trigeminal spinal nucleus is associated with orofacial thermal hyperalgesia in diabetic mice [21]. The increase in cPKCγ activity is also associated with diabetic embryonic dysmorphogenesis in rat [22]. However, few studies investigate the roles of the three specific PKC isoforms in the diabetic encephalopathy. In this study, we explored whether cPKCβII, cPKCγ, and nPKCε participated in the pathogenesis of type 1 diabetic encephalopathy induced by streptozocin (STZ) injection in mice.

Materials and Methods
2.1. Animals. C57BL/6 wild-type and cPKCγ knockout mice (male, 18-22 g) were purchased from Jackson Laboratory (Bar Harbor, Maine 04609, USA). Mice were housed, three per cage. The mice with free access to water and food were maintained in the Experimental Animal Center of Capital Medical University, the People's Republic of China. The experimental procedures were approved by the experimental animal ethics committee of Capital Medical University (permit number AEEI-2014-035) and conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Induction of Type 1 Diabetes Mellitus in Mice.
Animals were randomly divided into three groups, namely, the CON group (with citrate buffer treatment), T1DM group (with STZ treatment), and T1DM + cPKCγ group (cPKCγ knockout mice with STZ treatment). Following an overnight fast, T1DM mice were induced by low doses of freshly prepared STZ (50 mg/kg/day, i.p.), dissolved in a 0.1 M citrate buffer (pH 4.5), over five consecutive days. The control mice were only injected with citrate buffer (5 mL/kg/day, i.p.). The fasting blood glucose level was measured every week using a blood sample from the tail vein with the aid of a OneTouch® Ultra blood glucose meter (Milpitas, CA, USA). Mice with blood glucose levels over 11.1 mmol/L were considered diabetic and used for the study [23]. At 2 weeks and 8 weeks after STZ injection, the hippocampus, prefrontal cortex, and striatum of the mice in the CON and T1DM groups were prepared for the determination of PKC isoform expression levels and membrane translocation of PKC isoforms (n = 6 per group, resp.). At 8 weeks after STZ injection, the mice in the CON, T1DM, and T1DM + cPKCγ groups were tested in the Morris water maze (n = 12 per group, resp.).

Cytosolic and Membrane Fractions and Whole Tissue
Homogenate Preparation. As previously reported [24], the dissected samples were first homogenized at 4°C in buffer A [50 mM Tris-Cl, pH 7.5, containing 1 mM DTT, 2 mM EDTA, 2 mM EGTA, 50 mM potassium fluoride (KF), 5 mM sodium pyrophosphate, and a protease inhibitor mixture]. Homogenates were centrifuged at 30,000g for 30 min, and the supernatant was collected as cytosolic fractions. The pellet fractions (membrane fractions) were resuspended in buffer A containing 2% SDS before being sonicated and centrifuged again. These two fractions were analyzed for membrane translocation of PKC isoforms. To analyze PKC isoform expression, the whole tissue was homogenized and sonicated in RIPA buffer. The concentration of protein was analyzed as previously reported [25].
2.4. Western Blot. As previously reported [26], the samples (30 μg) were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (GE Healthcare, UK); the membrane was blocked with 10% nonfat milk for 1 h and probed with anti-cPKCβII (number sc-210, Santa Cruz Biotechnology, Santa Cruz, USA), cPKCγ (number sc-211, Santa Cruz Biotechnology, Santa Cruz, USA), and nPKCε (number sc-214, Santa Cruz Biotechnology, Santa Cruz, USA). The horseradish peroxidase-conjugated goat antirabbit (number 31460, ThermoFisher Scientific, Rockford, USA) and anti-mouse IgG (number 31430, ThermoFisher Scientific, Rockford, USA) were used as second antibodies at a 1 : 4000 dilution for 1 h at room temperature. Signals were visualized via an enhanced chemiluminescent reagent solution (chemiluminescent HRP substrate, Millipore Corporation, USA). To verify the equal loading of protein, the blots were reprobed with primary monoclonal antibodies against β-actin (number 60008-1-lg, Proteintech, Rosemont, USA) or Na + /K + ATPase (number 14418-1-AP, Proteintech, Rosemont, USA). The protein expression levels of PKC isoforms were calculated as 100% in the control group, and then the other groups were expressed as the percentage of the control group. Quantitative analysis for immunoblotting was done by Fusion Capt 16.15 software (Fusion FX6 XT, Vilber Lourmat, France).

Morris Water
Maze. At 8 weeks after STZ injection, spatial learning and memory were tested by the Morris water maze. Briefly, a custom-built water tank (122 cm diameter, 50 cm height) with a white nonreflective interior surface was filled with opaque water (19-22°C) containing nontoxic titanium white-colored dye. The experiment was performed in a room with low-light indirect lighting. Four different extramaze cues were fixed at four quartiles of the tank periphery ( Figure 1(e)). In the target quadrant (quadrant II), a platform was submerged 1 cm below the water surface. The mice were trained to find the hidden platform for 5 consecutive days, four trials of 90 seconds per day. In each trial, the mouse started from one of the four quadrants facing the wall of the pool and ended when the mouse climbed on the platform. If the mice did not find the platform in 90 s, they were guided to the platform. To test reference memory, the platform was removed on day 6 and the percentage of time that the mice spent in each quadrant was recorded. The visible platform tests (with the platform marked) were given on day 7 and day 8 to evaluate sensorimotor abilities and motivation of the animals. Swim paths were recorded on a CCD camera and analyzed using WaterMaze 3 Software. Average swimming speed was determined to exclude motor impairments.
2.6. Statistics Analysis. The values were presented as the mean ± SEM. Statistical analysis of Western blot was conducted by a one-way analysis of variance followed by all pairwise multiple comparison procedures using the Bonferroni test. The statistical analysis of the Morris water maze was conducted by a two-way analysis of variance followed by all pairwise multiple comparison procedures using the Bonferroni test. All results were regarded as at least p < 0 05 was considered statistically significant.

Biochemical
Characteristics of the Mice. At the beginning of the experiment, there was no significant difference among the groups in blood glucose and body weight. Following STZ injection, the mice developed DM rapidly with a mean blood glucose of 13.13 ± 4.74 mmol/L on day 3 after STZ injection, 18.87 ± 3.79 mmol/L at 2 weeks, and 26.7 ± 5.70 mmol/L at 8 weeks (Figure 1(a)). As shown in Figure 1(b), the body weight of the T1DM group increased (from 19.38 ± 2.03 g to 26.12 ± 2.36 g at 8 weeks after STZ injection) but much more slowly than the increase of the CON group (from 19.18 ± 1.54 g to 30.22 ± 1.43 g at 8 weeks after STZ injection). There was no significant difference in blood glucose and body weight between the cPKCγ +/+ diabetic mice and the scPKCγ −/− diabetic mice (Figures 1(a) and 1(b)).

Expression of PKC Isoforms in the Hippocampus, Prefrontal
Cortex, and Striatum of the T1DM Mice. In the next set of experiments, we observed whether diabetes mellitus affected the expression patterns of PKC isoforms in the hippocampus, prefrontal cortex, and striatum. As shown in Figures 2 and 3, we observed that the expression of cPKCβII, nPKCε, and cPKCγ did not change in the above brain regions at 2 or 8 weeks after STZ injection. These results indicated that chronic T1DM did not affect the expression of the three PKC isoforms in the hippocampus, prefrontal cortex, and striatum.

Membrane Translocation of PKC Isoforms in the Hippocampus, Prefrontal Cortex, and Striatum of the T1DM
Mice. The activation of PKC relates to a transition from the cytosolic autoinhibited form to the membrane-associated active form [27]. To figure out whether PKC isoforms are involved in type 1 diabetic encephalopathy, we observed the membrane translocation of PKC isoforms in the hippocampus, prefrontal cortex, and striatum of the T1DM mice.
As shown in Figures 4(c) and 5(c), chronic T1DM did not affect the membrane translocation of nPKCε in the hippocampus, prefrontal cortex, and striatum at 2 weeks and 8 weeks after STZ injection. The ratio of membranal cPKCβII to cytosolic cPKCβII was not significantly altered in the three brain regions at 2 weeks (Figure 4(a)) but dramatically decreased in the prefrontal cortex and hippocampus at 8 weeks ( Figure 5(a)). Moreover, chronic T1DM significantly reduced the ratio of membranal cPKCγ to cytosolic cPKCγ in the hippocampus at 2 weeks (Figure 4(b)) and in the prefrontal cortex, hippocampus, and striatum at 8 weeks ( Figure 5(b)).

Effects of cPKCγ on Spatial Learning and Memory of
Mice. To explore the role of cPKCγ in learning and memory, the Morris water maze test was used at 8 weeks after STZ injection. On day 5, the diabetic mice had a significantly increased latency to find the hidden platform compared to control mice (Figure 1(c)). Furthermore, the cPKCγdeficient diabetic mice took more time to find the hidden platform than the wild-type diabetic mice (Figure 1(c)).
On day 6, swim patterns indicated that control mice swam immediately towards the target area, where the platform used was placed, tried to find the platform, and swam back and forth. In contrast, diabetic mice swam in random circles throughout the pool (Figure 1(e)). In addition, the cPKCγ −/− diabetic mice performed worse than the wildtype diabetic mice (Figure 1(e)).
The percentage of the time spent swimming in the target quadrant (quadrant II) was significantly lower for diabetic mice than for control mice (Figure 1(d)). Moreover, cPKCγ −/− diabetic mice spent less time in the target quadrant than cPKCγ +/+ diabetic mice (Figure 1(d)), again indicating memory impairment in the cPKCγ knockout mice.
As both the vision and sensorimotor abilities of mice could affect the reliability of Morris water maze tests, the visible platform tests (with the platform marked) were given on day 7 and day 8. There were no significant differences in escape latency among groups (Figure 1(c)). As shown in Figure 1(f), no significant differences in average speed were observed among groups in 8 days. These results suggested that chronic T1DM could induce spatial learning and memory deficits of mice without affecting their visual and sensorimotor abilities and cPKCγ deficiency aggravated the spatial learning and memory impairment of T1DM mice.

Discussion
Several studies have reported that the expression of cPKCβII, cPKCγ, and nPKCε is altered by hyperglycemia. The expression of cPKCβII increases in the renal tissues of diabetic rats on 21 days after STZ injection [28]. The nPKCε level increases in the skeletal muscle of nutritionally induced diabetic rats in 3 weeks [29] and vessels of spontaneously diabetic rats [30]. And the expression of cPKCγ is also found to increase in the trigeminal spinal nucleus of T1DM mice on 14 days after STZ injection [21]. Interestingly, the expression of cPKCβII, cPKCγ, and nPKCε may not change in the cerebral cortex, but the levels of cPKCγ and nPKCε are decreased in the glio-pial tissue of T1DM rats at 16 weeks after STZ injection [31]. In this study, we explored the expression and activation of cPKCβII, cPKCγ, and nPKCε in the hippocampus, prefrontal cortex, and striatum, the regions important in the cognitive processes of learning, memory, and decision-making [32][33][34]. We observed that T1DM did not significantly change the protein expression of cPKCβII, cPKCγ, and nPKCε in the three brain regions at 2 weeks after STZ injection. Next, we prolonged the observation time. But we still did not find significant changes in the protein expression of cPKCβII, cPKCγ, and nPKCε at 8 weeks after STZ injection. Activation of PKC is associated with the translocation of enzymes from the cytosol to the membrane. The membrane translocation or activation of PKC isoforms is linked to the development of pathologies in diabetes. The acute blood glucose fluctuation significantly increased cPKCβII membrane translocation, which causes the apoptosis of vascular endothelial cell through increasing oxidative stress and induces impairment of insulin signaling in diabetic rats [11]. Activation of cPKCβII by hyperglycemia upregulates caspase 8-induced apoptosis in blood-brain barrier endothelial cells [35]. Activation of nPKCε, as a result of the accumulation of diacylglycerol, results in hepatic insulin resistance and nonalcoholic fatty liver disease in patients and type 2 diabetic mice [19,36]. Activation of cPKCγ by early STZ-induced diabetes causes the inhibition of lens gap junction activity in rats [37]. In this study, we found that T1DM mice exhibited spatial learning and memory impairment in the Morris water maze at 8 weeks after STZ injection. Meanwhile, T1DM failed to affect nPKCε membrane translocation in the hippocampus, prefrontal cortex, and striatum at 8 weeks. However, the translocation of cPKCβII and cPKCγ from the cytosol to the membrane was significantly decreased in the brain of T1DM mice at 8 weeks, indicating that they might be involved in type 1 diabetic encephalopathy.
It is well accepted that cPKCβII is essential for learning and memory formation [38,39]. The downregulation of cPKCβII in the hippocampus of offspring, especially in the CA3, CA1, and dentate gyrus, the regions important in the entorhinal-hippocampal trisynaptic circuit, is responsible for impaired learning and memory of prenatally stressed offspring [40]. The levels of cPKCβII decrease in the striatum and cortex of transgenic Huntington's disease mice, which is associated with impaired information storage and memory disorder [41]. The baseline asymmetry and lateralized changes of cPKCβII in the rat amygdale are associated with the cue and context in a classical fear conditioning paradigm [42]. And furthermore, the cPKCβII knockout mice also exhibit a loss of learning and suffer deficits in both cued and contextual fear conditioning [43].
cPKCγ is a neuron-specific isoform, and its activity is suggested to be related to spatial memory in aged rats [44,45]. Activation of cPKCγ in the CA1 region participates in postsynaptic plasticity associated with spatial experiences and learning [46]. Young rats, those with the best spatial memory, are those with the highest concentrations of cPKCγ in the particulate fraction. Conversely, aged rats with poor spatial memory increase hippocampal cPKCγ concentrations in soluble fraction in comparison with young rats, which indicates that the decrease in cPKCγ activity is correlated with memory impairment [47]. In addition, cPKCγ is found to participate in the reversal of learning and memory deficits by intranasal insulin treatment in AD mice [48]. In this study, we found that the decrease in cPKCγ activity induced by T1DM came earlier and occurred in more brain regions than cPKCβII, suggesting that cPKCγ played a bigger role in the diabetic encephalopathy. Therefore, we deleted the cPKCγ gene in mice and then found that the spatial learning and memory of cPKCγ −/− diabetic mice are worse than those of wild diabetic mice.
Elevated glucose level and impaired glucose tolerance can affect the spatial learning and memory, which is associated with hippocampal apoptosis [49][50][51][52]. But in this study, we found that cPKCγ did not affect blood glucose level, demonstrating that the effect of cPKCγ on cognitive function is irrelevant to blood glucose. PKC activators have been reported to improve cognitive function by restoring PKC signaling and downstream activity, including stimulation of synaptic plasticity and development [53], reduction in accumulation of neurotoxic amyloid β, regulation of tau protein hyperphosphorylation [38], and support of antiapoptotic processes [54]. Further studies about the mechanism how cPKCγ can enhance spatial memory performance in T1DM mice are required.

Conclusion
Our results indicate that the activation of cPKCβII and cPKCγ, not nPKCε, may be involved in the development of the diabetic encephalopathy in mice. cPKCγ can dramatically improve learning and memory performances impaired by diabetes mellitus in mice.

Conflicts of Interest
The authors declare that there are no conflicts of interest.


extfieldsofstudy: Chemistry Medicine

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Apoptosis induced by ursodeoxycholic acid in human melanoma cells through the mitochondrial pathway

Ursodeoxycholic acid (UDCA) is a type of hydrophilic bile acid extracted from animal bile with a wide range of biological functions. The present results demonstrated that UDCA could effectively inhibit the proliferation of two human melanoma cell line (M14 and A375) with time- and concentration-dependence. Following exposure to various concentrations of UDCA, M14 cells exhibited typical morphological changes and weaker ability of colony forming. Flow cytometry analysis demonstrated that UDCA could induce a decrease of mitochondrial membrane potential and an increase in reactive oxygen species (ROS) levels in M14 cells. The cell cycle was arrested in the G2/M phase, which was confirmed by the decrease of cyclin-dependent kinase 1 and cyclinB1 at the protein level. However, when M14 cells were treated with UDCA and Z-VAD-FMK (caspase inhibitor) synchronously, the apoptosis rate of the cells was reduced significantly. In addition, it was demonstrated that UDCA induced apoptosis of human melanoma M14 cells through the ROS-triggered mitochondrial-associated pathway, which was indicated by the increased expression of cleaved-caspase-3, cleaved-caspase-9, apoptotic protease activating factor-1, cleaved-poly (ADP-ribose) polymerase 1 and the elevation of B cell lymphoma-2 (Bcl-2) associated X protein/Bcl-2 ratio associated with apoptosis. Therefore, UDCA may be a potential drug for the treatment of human melanoma.

Introduction
Ursodeoxycholic acid (UDCA) is a type of hydrophilic bile acid that is produced by intestinal bacteria and accounts for only 4% of total bile acids in humans (1). UDCA has been reported to reduce the concentration of eosinophilic bile acids in bile acids and serum, as well as tumor necrosis factor-α in chronic cholestasis (2). Unlike toxic hydrophobic bile acids acting as carcinogens in various organs (the liver, colon and gastrointestinal tract), hydrophilic bile acids have long been considered as tumor suppressors for hepatocellular and colon cancer, as well as a reducer of cholestatic liver diseases (3)(4)(5)(6)(7). However, it remains unclear whether UDCA could suppress the growth of human melanoma.
Melanoma is the deadliest form of skin cancer and the incidence of melanoma in the population has increased steadily in recent years: According to the World Health Organization, there are 132,000 cases of cutaneous melanoma worldwide each year (8), and according to statistics from the American Cancer Society, ~9,730 people succumbed to melanoma in the United States in 2017 (9,10). The high mortality in melanoma patients has prompted novel research on therapies and drugs that increase efficacy, but the long-term survival of melanoma patients remains poor (11,12). Therefore, there is an urgent need to identify novel therapeutic agents or novel therapeutic strategies to improve the outcome of patients with advanced melanoma.
Apoptosis serves an important role in the normal development and differentiation of multicellular organisms (13,14). Activation of apoptosis in cancer cells may contribute to the elimination of cancer cells. The extrinsic and intrinsic pathways are the two major pathways regulating apoptosis, which are also known as death receptor pathways and mitochondrial pathways (13,15). Mitochondria are two-layered membrane organelles that are present in cells, and are the main organelles that maintain homeostasis within cells and the main site for aerobic respiration of cells (16). The destruction of mitochondria is an important feature of apoptosis. Cytoplasmic contraction, extensive plasma membrane blistering and nuclear condensation are the morphological features of apoptosis, which develops in a caspase-dependent or caspase-independent manner (17,18). In addition, the ratio of anti-apoptosis and pro-apoptotic mitochondrial proteins B cell lymphoma-2 (Bcl-2) associated X protein (Bax)/Bcl-2 determine the susceptibility of cells to apoptosis (19).
In the present study, UDCA suppressed the growth of M14 cells and induced apoptosis through mitochondrial-associated apoptosis pathways, indicating that UDCA may become a potential agent for the treatment of human melanoma.
Cell viability assay. Briefly, M14 cells were seeded at a density of 5x10 3 cells/well in 96-well microplates at 37˚C and 5% CO 2 for 24 h, and then the cells were treated with UDCA at different concentrations (0, 50, 100, 150, 200, 250 and 300 µg/ml) at 37˚C for 24, 48 and 72 h. Subsequently, 20 µl MTT solution was added to each well followed by incubation at 37˚C for 4 h. Finally, the culture solution was discarded and 150 µl DMSO was added to each well. The absorbance value was detected at a wavelength of 490 nm using a microplate reader.
Observation of cell morphology changes. A total of 3x10 5 M14 cells/well were seeded onto the 6-well coverslips and allowed to adhere at 37˚C and 5% CO 2 for 12 h prior to treatment with different concentrations of UDCA (0, 100, 200 and 300 µg/ml) at 37˚C for 48 h. Subsequently, cells were washed with PBS three times and stained with AO/EB at room temperature for 10 min. Finally, the cells were washed twice followed by observation under fluorescence microscopy (magnification, x200). In addition, M14 cells were washed with PBS, fixed with methanol at room temperature for 10 min, stained with Hoechst 33258 at room temperature for 7 min and observed under fluorescence microscopy (magnification, x200).
Cell colony formation assay. M14 cells were seeded into 6-cm plates (500 cells/plate) and allowed to adhere at 37˚C and 5% CO 2 for 12 h. The old medium was then discarded and different concentrations of UDCA (0, 100 200, and 300 µg/ml) was added at 37˚C and 5% CO 2 for 48 h. Subsequently, the medium containing UDCA was discarded, and cells were allowed to culture in new media for two weeks. Finally, the cells were fixed with anhydrous ethanol at room temperature for 15 min followed by washing with PBS twice, stained with Giemsa at room temperature for 15 min, washed with PBS twice, photographed and colonies were counted manually.
Cell migration assay. M14 cells were cultured at 37˚C in 5% CO 2 (v/v) until the cells covered the entire bottom of the 6-well plate. The old medium was discarded and a small 10-µl white pipette was used to draw an artificial wound area at the bottom of the dish. Following treatment with different concentrations of UDCA (0, 100, 200, and 300 µg/ml) at 37˚C and 5% CO 2 for 48 h, the cells were washed, then fixed in pure methanol at room temperature for 10 min. The wounds were photographed under inverted ordinary phase-contrast microscopy (TE2000-U; Nikon Corporation, Tokyo, Japan) equipped with NIS-Elements (Nikon Corporation; magnification, x200).
Cell cycle distribution analysis. A total of 3x10 5 M14 cells/well were seeded onto 6-well plates and allowed to adhere at 37˚C and 5% CO 2 for 12 h and following treatment with different concentrations of UDCA (0, 100, 200, and 300 µg/ml) for 48 h, cells were collected by centrifuging at 1,500 x g at 4˚C for 10 min and the precipitations were washed once with PBS. The cells were fixed with a pre-cooled ethanol-PBS mixture (7:3, v/v) at 4˚C for 60 min and washed twice with PBS. Finally, 10 mg/ml RNase and 1 mg/ml PI was added followed by incubating at 37˚C for 30 min in the dark. Following filtration of the cells with a 300-mesh sieve, the cells were detected by flow cytometry and processed using the ModFit LT 3.3 (Verity Software House, Inc., Topsham, ME, USA) analysis software.
Cell apoptosis rate analysis. The apoptosis rate was measured using the Annexin V-FITC/PI Apoptosis Detection kit. M14 cells were seeded using DMEM at a density of 5x10 3 cells/well in 6-well microplates at 37˚C and 5% CO 2 for 24 h, and then the cells were treated with UDCA at different concentrations (0, 100, 200, and 300 µg/ml) at 37˚C for 48 h., cells were collected and then washed once with PBS; 195 µl binding buffer (1X) and 5 µl Annexin V-FITC was then added to each sample, which were then incubated at 37˚C for 15 min. Following centrifugation at 1,500 x g at 4˚C for 5 min, 200 µl binding buffer (1X) was added to each tube to wash; subsequently, 190 µl binding buffer (1X) and 10 µl PI solution was added to each tube to resuspend the cells, and incubated at room temperature for 15 min. The cell apoptosis rate was measured by a FC500 flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) within 4 h, and the data was analyzed using FCS analysis software (Beckman Coulter, Inc.).
Mitochondrial membrane potential (ΔΨi) analysis. The mitochondrial membrane potential changes in treated cells which were seeded using DMEM at a density of 5x10 3 cells/well in 96-well microplates at 37˚C and 5% CO 2 for 24 h and following by UDCA treatment (0, 100, 200, and 300 µg/ml) were measured using a mitochondrial membrane potential assay kit (JC-1). The cells were washed with PBS once, and 500 µl culture medium was added to each tube to resuspend the cells. An equal volume of JC-1 working fluid was added and the tube was inverted several times. The cells were incubated at 37˚C and 5% CO 2 (v/v) for 20 min and washed twice with JC-1 staining buffer (1X). The cells were resuspended in JC-1 staining buffer (1X) at room temperature for 5 min and the intracellular mitochondrial membrane potential was detected using a FC500 flow cytometer (Beckman Coulter, Inc.) and the data was analyzed using FCS analysis software.
Reactive oxygen species (ROS) analysis. A total of 3x10 5 M14 cells /well were seeded onto the 6-well plates and allowed to adhere at 37˚C and 5% CO 2 for 12 h, following treatment with different concentrations of UDCA (0, 100, 200, and 300 µg/ml) for 48 h, M14 cells were centrifuged at 3,000 x g at 4˚C for 5 min, and the pellet was washed once with PBS. Subsequently, 500 µl 10 mmol/l DCFH-DA was added to each tube, and the cells were resuspended and incubated at 37˚C in 5% CO 2 (v/v) for 20 min. Cells were then centrifuged at 1,500 x g at 4˚C for 5 min and the supernatant was discarded. Finally, the cells were filtered through a 300-mesh sieve and active oxygen content was determined by a FC500 flow cytometer (Beckman Coulter, Inc.) and the data was analyzed using WinMDI 2.9 (The Scripps Research Institute, La Jolla, CA, USA).
Z-VAD-FMK treatment. M14 cells were plated in 6-well plates as described above, but treated with UDCA (300 µg/ml) and Z-VAD-FMK (40 µM) alone or in combination. Then the morphological changes of these cells were observed and changes in apoptosis rate were detected as described above.
Western blot analysis. M14 cells were plated in 6-well plates as described above following treating M14 cells with UDCA (0, 100, 200, and 300 µg/ml) at 37˚C and 5% CO 2 for 48 h, the cells were collected and washed twice. Proteins were extracted with RIPA buffer and centrifuged at 20,000 x g for 30 min at 4˚C. Total protein concentration was determined using the bicinchoninic acid assay protein assay kit (Beyotime Institute of Biotechnology). Subsequently, the same amount of protein lysates (50 ug/lane) added with loading buffer were separated by 10% SDS-PAGE, which were then transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% fat-free milk at 4˚C for 1 h. Soon afterwards, the membrane was incubated with the specific primary antibodies (diluted with ddH 2 O to 1:1,000) at 4˚C overnight, and then proteins were visualized with horseradish peroxidase-coupled secondary antibodies (1:10,000) at room temperature for 1 h. Finally, the results were detected using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).
Observation of mitochondria with transmission electron microscopy (TEM). A total of 3x10 5 M14 cells/well were seeded onto the 6-well plates with DMEM and allowed to adhere at 37˚C and 5% CO 2 for 12 h, following 48 h of UDCA treatment (0, 100, 200, and 300 µg/ml) of M14 cells, cells were collected and washed, and then fixed in 4% formaldehyde at 4˚C for 2-3 h. Next, the specimens were cut into 4-5 8-mm 3 samples. Cell pellets were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4˚C for 2 h followed by washing with PBS, fixed with 1% osmium tetroxide at 4˚C for 1 h and then dehydrated with alcohol and acetone. Samples were embedded at room temperature overnight in spur-resin, then heated to 70˚C for 9 h in capsules. Finally, the samples were cut into 70-nm-thick sections using an ultramicrotome, and the specimens were double-stained with uranyl acetate and lead citrate for 10 min each. The sample was observed and photographed using a transmission electron microscope (magnification, x1,000).
Statistical analysis. All presented data were performed at least in triplicate. Data are presented the as mean + standard deviation. Differences between two groups were analyzed using Student's t-test, and one-way analysis of variance followed by Dunnett's multiple comparisons test was applied for more than two groups. P<0.05 was considered to indicate a statistically significant difference.   To explore whether UDCA could affect the colony-forming ability of M14 cells, 500 M14 cells were seeded into 6-cm plates and then treated with UDCA. As demonstrated in Fig. 1E, when the concentration of UDCA was 100 µg/ml, the colony forming ability of M14 cells did not change markedly. However, when the concentration of UDCA was 200 and 300 µg/ml, the colony forming ability of M14 cells was markedly decreased. In addition, UDCA could affect the migration ability of M14 cells, as demonstrated in Fig. 2A.

UDCA inhibits the proliferation of two human melanoma
To confirm whether M14 cells undergo morphological changes following UDCA treatment, their morphological characteristics were observed with an optical inverted phase contrast microscope. As demonstrated in Fig. 2B, M14 cells became smaller and more rounded following UDCA treatment compared with the control group. Following Hoechst 33258 staining, the UDCA-treated cells exhibited brighter blue light than that of the control cells, which indicated pyknotic and deep-dyed nuclei. With AO/EB staining, some of the UDCA-treated M14 cells emitted orange and red fluorescence whose size was smaller compared with the control group.
In addition, UDCA significantly inhibited the proliferation of A375 cells and induced morphological changes following UDCA treatment as demonstrated in Fig. 3A and B. In Fig. 3A, with different concentrations of UDCA (250 and 300 µg/ml) for 48 h, the inhibition rates were 20.61 and 34.47% compared with the 0 µg/ml group respectively. In Fig. 3B, A375 cells became smaller and more rounded following UDCA treatment compared with the control group. Following Hoechst 33258 and AO/EB staining, A375 cells exhibited typical morphological changes in apoptosis as in M14 cells. However, UDCA exhibited more evident toxicity in M14 compared with A375 cells at the same concentration and UDCA did not markedly affect the migration of A375 cells (Fig. 3C).
These results indicated that M14 cells exhibited marked apoptosis-associated morphological features and the colony forming ability was decreased following UDCA treatment. UDCA inhibits the migration of M14 cells. Wound healing was used to determine whether UDCA could affect the migration ability of M14 cells. The results are presented in Fig. 2A. Following culture for 96 h, cells in the control group quickly migrated to the scratches and the scratches were notably compressed. In the 100 µg/ml group, wounds in the UDCA-treated group were similar to those in the control group; the wounds in the 200 µg/ml group were larger than those in the first two groups and the wounds in the 300 µg/ml group were wider still. However, UDCA did not markedly inhibit the migration of A375 cells at the same concentration in M14 cells (Fig. 3C).

UDCA induces cell cycle arrest in M14 cells.
To further explore the role of UDCA in inhibiting the proliferation of M14 cells, flow cytometry was used to detect the cell cycle distribution of M14 cells under different concentrations of UDCA (Fig. 4), by detecting the DNA content of cells (20). M14 cells were stained with PI following 48 h of UDCA treatment to quantify the DNA content of cells. As revealed in Fig. 4A

UDCA induces apoptosis in M14 cells.
To distinguish the normal, early and late apoptotic cells, flow cytometric analysis was conducted by double staining. In Fig. 4B and D, the results indicated that the apoptosis rate induced by UDCA presented a dose-dependence from 0-300 µg/ml. Total proportion of apoptotic cells increased to 12.18% of 300 µg/ml-treatment group from the 1.65% of the control. These results indicated that UDCA inhibits the growth of M14 cells by inducing apoptosis via activation of caspases.
To further confirm the effect of UDCA on M14 cells by inducing apoptosis, AO/EB and Hoechst 33258 staining were performed to observe the change of cell morphologies. AO/EB staining could distinguish apoptotic and necrotic cells with different fluorescence. As demonstrated in Fig. 2B, the control group exhibited a normal morphology and a pale green fluorescence. However, when cells were treated with different concentrations of UDCA, the number of cells gradually decreased and some cells emitted bright green and red fluorescence. When UDCA-treated M14 cells were stained with Hoechst 33258, typical apoptotic features were detected including chromatin condensation and the destructive fragmentation of nuclei as demonstrated in Fig. 2B.
UDCA triggers the generation of ROS in M14 cells. ROS are a by-product of the aerobic metabolism of organisms (21). It has previously been demonstrated that active oxygen is closely associated with tumor formation, cell cycle and apoptosis (22). To detect whether UDCA could induce increased intracellular ROS levels in M14 cells, the intracellular ROS level was examined using flow cytometry (Fig. 5). The mean value of ROS was markedly elevated with the increase of UDCA concentration as demonstrated in Fig. 5A and C. The ROS generation in the 300 µg/ml-treatment group increased by 65.27% compared with the control group, which suggested that the increase of ROS was one of the main factors of apoptosis.

UDCA induces mitochondrial dysfunction in M14 cells.
Flow cytometry was used to examine the quantification of ΔΨm in M14 cells following treatment with UDCA. The decrease in ΔΨm is a hallmark event in early stages of apoptosis (23,24). JC-1, a fluorescent probe widely used to detect mitochondrial membrane potential, is able to detect cells, tissues and purified ΔΨm (25). When the ΔΨm is high, JC-1 in the form of a polymer, accumulates in the matrix of mitochondria, and produces red fluorescence; when the ΔΨm decreases, JC-1 flows out of the mitochondrial matrix in the form of a monomer, producing green fluorescence (26). Therefore, the degree of mitochondrial depolarization could be quantified by calculating the ratio of red-green fluorescence. As demonstrated in Fig. 5B and D, following M14 cells exposed to UDCA for 48 h, there was a significant increase of the mitochondrial depolarization from 1.5 to 27.5% at 300 µg/ml compared with the control. The results indicated that each concentration of UDCA could effectively reduce the ΔΨm with dose-dependence.

Observation of mitochondrial morphology in M14 cells treated with UDCA using TEM.
In the present study, UDCA was used to induce melanoma cell apoptosis. As demonstrated in Fig. 6A, mitochondrial morphological changes mainly included two stages: When the concentration was 100 µg/ml, the number of mitochondria increased, the distribution was not uniform and some mitochondria began to exhibit dense material deposition; when the concentration was 200 µg/ml, mitochondria swelled, and the structure of mitochondria disappeared, leaving only a single layer of film. Nuclear chromatin condensation appeared at 300 µg/ml and adhered to the nuclear membrane to form a wavy curve profile, which was typical of apoptotic cells (27). Therefore, during the entire process of apoptosis in the present study, the changes in mitochondria preceded that in the nucleus.
Changes in M14 apoptosis rate treated with UDCA and caspase inhibitors (Z-VAD-FMK). The ratio of the apoptotic cells decreased when M14 cells were synchronously treated with UDCA and Z-VAD-FMK ( Fig. 6B and C). These morphological observations on apoptosis were consistent with the results of the flow cytometric analysis. The UDCA-induced M14 cell apoptosis ratio could be reduced by the Z-VAD-FMK (40 µM) from 12.18 to 6.35% (Fig. 6C and D).
Western blotting. The expression of CDK1 and cyclinB1 at the proteins level were decreased distinctly as demonstrated in Fig. 7A, which was consistent with the results of the flow cytometry analysis in Fig. 4A and C. In addition, western blotting results demonstrated that the expression of MMP-2 and MMP-9 at the protein levels were markedly decreased following UDCA treatment compared with the 0 µg/ml-UDCA group (Fig. 7B), which was consistent with the results of wound healing analysis in Fig. 2A. PARP1 protein is a marker of apoptosis, which was activated immediately by cleaved-caspase. The activation of p53, the release of cytochrome c and the cleavage of caspase-3 and PARP1 were increased in a dose-dependent manner as   Fig. 7C-E. Cytochrome c in cytoplasm gradually increased with UDCA treatment, which demonstrated that cytochrome c was gradually released from the mitochondria into cytoplasm (Fig. 7E). In addition, the ratio of Bax/Bcl-2 serves an important role in regulating apoptosis, which was demonstrated in Fig. 7A. Finally, the results of all protein molecule expression changes are summarized in Fig. 7F.

Discussion
The efficacy of UDCA in primary biliary cirrhosis has been identified previously (28). UDCA is able to improve a series of biochemical and histological parameters (prothrombin time, alkaline phosphatase, alanine transaminase, fatigue, hepatomegaly or splenomegaly) induced by primary biliary cirrhosis, thereby prolonging the patient's survival (29). It has previously been reported that UDCA could effectively induce apoptosis in liver cancer, gastric cancer and colon cancer cell lines (7,(30)(31)(32). However, to the best of our knowledge the role of UDCA in human melanoma has not yet been explored. The present study investigated the effect of UDCA on the proliferation and apoptosis of human melanoma cell lines to observe its mechanism of the effects. The present data demonstrated that UDCA could significantly inhibit the growth of human melanoma M14 and A375 cells, but the inhibitory effect on LO2 human normal liver cells and the human keratinocyte cells HaCaT in the same concentration for comparison was not obvious. However, a number of other chemotherapeutic drugs currently in use have many side effects, such as ipilimumab, nivolumab and pembrolizumab (11,12,33).
LO2 is a human normal liver cell. In the present study, LO2 cells were selected as the cultured cell representing tissue from the biggest digestive gland, the liver, where 90% of drugs are metabolized by cytochrome P450 enzymes (34) with the majority of them producing efficacy or toxicity afterwards (35). In addition, the liver is very sensitive to xenobiotics and is subject to damage by drug doses several times lower than that which damages other organ tissues (36). In addition, the results of the MTT assay for detecting the inhibitory effect of UDCA on HaCaT cells at different time points demonstrated that UDCA was specific for the inhibition of M14 cells.
To the best of our knowledge, the present study is the first to explore the mechanism of UDCA-induced apoptosis in M14 cells. It has been reported that there are two primary methods of cell death: Apoptosis and cell necrosis. The present results demonstrated that UDCA could significantly inhibit the proliferation of M14 and A375 cells but caused little toxicity in LO2 and HaCaT cells. In addition, UDCA had more evident toxicity in M14 compared with A375 cells at the same concentration. Therefore, the mechanism of UDCA induced apoptosis in M14.
The present experiments further confirmed that UDCA could inhibit cell growth by inducing cell cycle arrest and cell apoptosis in M14 cells. In cancer, controlling cell cycle is an effective way to inhibit tumor growth. The present results demonstrated that UDCA arrested the cell cycle of M14 cells in the G2/M phase by downregulating the key factor CDK1 and cyclinB1 proteins (37). These data indicated that the periodic block of M14 by UDCA was an important signaling pathway for its anti-proliferative effect. The present Annexin V-FITC/PI staining results clearly demonstrated that the rate of apoptosis induced by UDCA was dose-dependent. At the same time, in order to further confirm that UDCA could promote cell apoptosis, a series of morphological changes in M14 cells were detected. A series of morphological changes including a change from the shuttle-type to irregular polygon-or round-shaped, chromosome condensation, deepening of staining, nuclear debris and apoptotic bodies, was consistent with the results of flow cytometry analysis. These morphological changes are also typical features of apoptosis (38,39), thus UDCA might induce M14 cell death by apoptosis.
Apoptosis comprises two pathways, including the mitochondria-mediated inward apoptotic pathway and the death receptor-mediated external apoptotic pathway (39). Mitochondrial dysfunction is the initial step in the intrinsic apoptotic pathway (40). In addition, ΔΨm arises from respiration. The electron transport chain pumping protons out of the inner membrane is a key step in the production of ATP (41). Therefore, changes in ΔΨm have been considered as one of the hallmark events of early apoptosis. In mitochondrial endogenous apoptotic pathways, cells are subjected to external stimuli resulting in changes in ΔΨm, which in turn leads to an increase in apoptosis-associated proteins and cytochrome c release-related proteins (19). Finally, cytochrome c binds to Apaf-1 and this formed protein complex initiates apoptosis by activating downstream caspase. In the present study, the results of flow cytometry indicated that UDCA at all concentrations could effectively reduce ΔΨm. With the increased concentration of UDCA, the degree of mitochondrial depolarization increased gradually. Changes in the ΔΨm could lead to changes in the conformation of the permeability transition pore, which in turn led to changes in the mitochondrial structure. This structural damage induced the release of cytochrome c.
ROS are a by-product of the aerobic metabolism of organisms. Previous studies have demonstrated that active oxygen is closely associated with tumor formation, cell cycle and apoptosis. The present results demonstrated that intracellular ROS levels were significantly increased following UDCA treatment. It was previously indicated that intracellular ROS accumulation is strongly associated with apoptosis (42). Once the accumulation of ROS in the cell reaches a certain level, it will lead to mitochondrial dysfunction and p53 activation which serves a vital role in cell apoptosis (43). The present western blotting results demonstrated that p53 protein was significantly upregulated with increasing UDCA concentration. This indicated that the accumulation of ROS induced the activation of p53 and led to the apoptosis of M14 cells. It has been reported that Bax and Bcl-2 proteins are important members of Bcl-2 family proteins, of which the former has a pro-apoptotic effect, and the latter has an inhibitory effect on apoptosis (44). In addition, Bax protein is an important transcriptional target of p53 protein (45). In addition, Bax protein promotes the release of cytochrome c from mitochondria into cytosol to active caspase-3 and finally, the cleavage of caspase-3 leads to cleavage of PARP1, which leads to cell apoptosis (40). In the present study, the protein expression level of Bax was not markedly amended, but the protein expression level of Bcl-2 had decreased, and the cleavage of PARP1 was elevated significantly, which demonstrated that UDCA regulated M14 apoptosis via elevating the ratio of Bax/Bcl-2 proteins and caused the elevation of p53 which triggered ROS production. Furthermore, electron microscopic observations revealed that the mitochondrial morphology in M14 cells was markedly changed following treatment with UDCA.
In conclusion, UDCA is a novel active compound that could arrest the M14 cells at the G2/M phase and induce apoptosis through ROS-triggered mitochondrial-associated pathways. Therefore, UDCA might become a potential drug for the treatment of melanoma. However, a limitation of the present study is that it was mainly focused on the effect of UDCA in M14 cells; therefore, further studies with A375 and other melanoma cell lines are required.


extfieldsofstudy: Biology Medicine

text:
Circular RNAs in hepatocellular carcinoma: Functions and implications

Abstract At present, as hotspot members of the noncoding RNA network, circular RNAs (circRNAs) with distinct properties and diverse pathophysiological functions are being increasingly delineated. CircRNAs play roles at the epigenetic, transcriptional and posttranscriptional regulatory levels. Major studies have focused on their functions as efficient microRNA sponges. The validated number of endogenous circRNAs involved in hepatocellular carcinoma (HCC) continues to increase. Altered circRNA expression is associated with HCC occurrence, invasion, and metastasis. Moreover, the aberrant expression of circRNAs is also significantly related to HCC tumor stage, size, differentiation and metastasis. Because they are exceptionally stable, highly conserved and have tissue‐specific expression patterns, some circRNAs, including hsa_circ_0004018, hsa_circ_0003570, and hsa_circ_0005075, may be potential markers for the diagnosis of HCC. We herein summarize the current knowledge of HCC‐associated circRNAs and present their implications for carcinogenesis and their potential value as diagnostic and prognostic biomarkers. Finally, we discuss the future directions of studies on HCC‐associated circRNAs.

them the properties of exonuclease resistance, long half-life and inherent stability. Now, an increasing number of studies has demonstrated that circRNAs play important biological roles in the normal development of tissues or organs and in the occurrence and pathogenesis of diseases. [1][2][3] Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy derived from intricate genetic and epigenetic alterations and is the second leading cause of cancer-related death worldwide. When diagnosed at the early stage, HCC patients can achieve marked advances in life expectancy; however, when diagnosed at the advanced stage with metastasis, patients have dismal prognoses, even if they are treated with multikinase inhibitor sorafenib or regorafenib. 4 The challenges confronting hepatologists worldwide are mainly focused on how to screen HCC patients at the earlier stage and thereby perform timely curative procedures, such as radical primary resection, ablation or liver transplantation. Therefore, exploring the molecular mechanism and identifying valuable markers of HCC are extremely important.

| BIOGENESIS OF CIRCRNAS
CircRNAs are formed through lariat-circulating or backsplicing events from all regions of the genome and are derived mostly from exons; however, in rare cases, circRNAs are derived from intergenic, intronic regions, antisense or untranslated region (UTR) regions. The terms circRNA, EIciRNA and ciRNA are used to describe circRNAs originating from the exon, exon and intron, and intron, respectively ( Figure 1). CircRNAs can be found in most subcellular compartments, but the majority are localized predominantly in the cytoplasm. [1][2][3]5 CircRNA formation is influenced by several sequence features, such as intron length, exon length, repetitive sequences, and RNA-binding proteins (RBPs), including quaking (QKI), adenosine deaminases that act on RNA (ADAR1), NF90/NF110, heterogeneous nuclear ribonucleoprotein L (HNRNPL) and muscleblind (MBL/ MBNL1). [6][7][8][9][10][11] Recently, based on RNA interference screening, Liang et al 12 revealed that the expression ratio of linear to circular RNA is modulated by many core spliceosomal and transcription termination factors.
How are circRNAs degraded or cleared? Scientists have recently focused on exosomes. Exosomes, which are doublemembrane vehicles containing different functional molecules, such as proteins, microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circRNAs, may mediate cell-cell communication in a paracrine or endocrine manner 13 (Figure 2). The expulsion of circRNAs through exosomal release might be an effective way to clear or degrade circRNAs. 14

| Regulation of transcription
Intron-containing circRNAs, such as ciRNAs and EIciRNAs, are abundant in the nucleus. How ciRNAs and EIciRNAs are restricted to the nucleus remains vague. These circRNAs may regulate the expression of their host genes in either a cis-acting manner or a trans-acting manner (Figure 2A). Some ciRNAs, such as ci-ankrd52 and ci-sirt7, can gather at the transcriptional sites of host genes, interact with the RNA polymerase II (Pol II) complex, and act as positive regulators of parental gene transcription. 15 Li et al 16 found that EIciEIF3J and EIciPAIP2 can interact with the Pol II complex and U1 small nuclear ribonucleoproteins (snRNP) and thereby increase Eukaryotic translation initiation factor 3 subunit J (EIF3J) and Polyadenylate-binding protein-interacting protein 2 (PAIP2) transcription in a cis-acting manner.

| MiRNA sequestration
CircRNAs harbor well-conserved canonical miRNA response elements (MREs), suggesting that some circRNAs F I G U R E 1 The biogenesis of circular RNA (circRNA). Circular RNAs are generated from lariat-driven circulation or back-splicing events between the splice donor site of a downstream exon and the splice acceptor site of an upstream exon act as miRNA sequesters or sponges. [17][18][19][20] A portion of cir-cRNAs are thought to negatively regulate the levels of miR-NAs and increase or decrease the expression levels of their corresponding targeted genes ( Figure 2B). As a representative circRNA, cerebellar degeneration-related protein 1 antisense (CDR1as) is the sponge of miR-7 in the embryonic zebrafish and has been reported to be related to brain dysfunction and many cancers, such as HCC and gastric cancer. [21][22][23][24] Accordingly, an increasing number of studies has revealed that the circRNA-miRNA-target gene regulatory network might have profound implications for understanding diseases, especially cancers. 25,26 For example, a recent study shows that circ-ZNF609 acts as an endogenous miR-615-5p sponge to inhibit miR-615-5p activity, increasing myocyte-specific enhancer factor 2A (MEF2A) expression and ameliorating vascular endothelial dysfunction. 27 Zhong et al 28 found that circ-MYLK is not only related to the stage and grade of the cancer but can also work with miR-29a and relieve the suppression of target vascular endothelial growth factor A (VEGFA), a key member of the VEGFA/VEGFR2/ Ras/ERK signaling pathway. Increased circ-UBAP2 promotes cancer cell growth and inhibits apoptosis both in vitro and in vivo by sequestering miR-143, thus enhancing its target antiapoptotic Bcl-2 expression. 29 Liang et al 30 disclosed that circ-ABCB10 can sponge miR-1271, increasing proliferation and decreasing apoptosis. Hsa_circ_000984 derived from a cell division protein kinase (CDK6) may act as a competing endogenous RNA (ceRNA) by sequestering miR-106b. 31 Circ-HIPK2 derived from HIPK2 gene exon 2 can affect the distinct roles of its target in cell autophagy by sponging miR-124 (miR124-2HG), thus regulating sigma nonopioid intracellular receptor 1 (SIGMAR1/OPRS1) expression in astrocyte cells. 32 However, circ-HIPK3 derived from HIPK3 gene exon 2 can abundantly sponge miR-558 F I G U R E 2 Function of circRNAs. A, ciRNAs interact with RNA Pol II, and EIciRNAs associate with RNA Pol II and U1 snRNP and thereby enhance the transcription of their parental genes. B, Most circRNAs can act as miRNA sponges or sequesterers. C, CircRNAs with IRES elements might be used as translational templates. D, CircRNAs with RBP motifs may function as sponges or decoys for proteins and thereby regulate their activity. E, CircRNAs with binding motifs for an enzyme and its substrate may function as scaffolds facilitating co-localization and reaction kinetics. F, Exosomes containing circRNAs are secreted from cells into blood vessels or mediate cell-cell communication. Abbreviations: IRES: Internal ribosome entry site, TAMs: tumor associated macrophages, TAFs: tumor associated fibroblasts to suppress the expression of heparanase (HPSE) in bladder cancer and can sponge endogenous miR-30a-3p in diabetes mellitus-associated retinal vascular dysfunction. 33,34

| Templates for translation
As most circRNAs originate from exons, are located in the cytoplasm, might contain internal ribosome entry site (IRES), and include an open reading frame (ORF), they may be efficiently translated 35,36 (Figure 2C). Yang et al validated that some cir-cRNAs are rich in N6-methyladenosine (m6A) motifs that enable translation initiation. This study revealed that thousands of endogenous circRNAs might have translation ability. 37 Yang et al 38 validated that circ-FBXW7 contains an ORF driven by IRES and translates into a novel 21-kDa protein (termed FBXW7-185aa). Legnini et al 39 identified an ORF-containing circ-ZNF609 translated into a protein in a splicing-dependent/ cap-independent manner in human and murine myoblasts.

| CircRNA-protein interaction
CircRNAs may be used to bind, store, or sequester protein molecules. Most circRNAs may interact with RBPs ( Figure 2D). With a high density of binding sites for a single or multiple RBP, some circRNAs may function as protein sponges or decoys. EIciRNAs and ciRNAs promote the transcription of their parental genes by interacting with host U1 snRNP and/or RNA Polymerase II. The best experimentally supported example of a circRNA interacting with a protein is CDR1as, which is associated with Argonaute (AGO) proteins in a miR-7-dependent manner. 21,40 Coon et al 6 validated that the circRNA abundance decreases upon QKI knockdown and fluctuates during the human epithelial-mesenchymal transition (EMT) process. The circRNA derived from the MBL locus (circ-MBL) harbors binding sites for the MBL protein itself and prevents MBL protein from binding to other targets when tethered to the circ-MBL. 8 In additional, the circRNA derived from the PABPN1 locus (circ-PABPN1) harbors binding sites for ELAV-like protein 1 (HuR) and hence prevents HuR from binding to PABPN1 mRNA and lowers its translation. 41 In contrast to the vast majority of exon-derived circRNAs that are located in the cytoplasm, circ-Amotl1 (hsa_circ_0004214) tends to interact with and stabilize oncogene C-MYC in the nucleus. Hsa_circ_0004214 enhances C-MYC binding to the promoters of hypoxia-inducing factor (HIF)-1α, cell division cycle 25 homolog A (Cdc25a), ETS domain-containing protein (ELK1), and JUN, thus promoting cell proliferation, invasion and colony formation. 42

| Scaffold for enzymes
Those circRNAs that harbor binding domains for enzymes and their substrates are likely to function as scaffolds to connect two or more proteins ( Figure 2E). To date, circ-Foxo3 is the best example of displaying a variety of tertiary structures in various cell/tissue environments. 43 In the mouse fibroblast NIH3T3 cell line, circ-Foxo3 can bind to cyclin-dependent kinase inhibitor 1 (p21) and cyclin-dependent kinase 2 (CDK2), and this binding represses cell cycle entry. 44 Mouse double minute 2 homolog (MDM2) is an important negative regulator of the p53 tumor suppressor, and MDM2 protein functions as both an E3 ubiquitin ligase that recognizes the N-terminal transactivation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. Circ-Foxo3 can bind to MDM2 and p53, promote MDM2-induced p53 ubiquitination and subsequent degradation, and thus induce tumor cell apoptosis. 45 In addition, circ-Foxo3 can bind to stress-related proteins and transcription factors, such as DNA-binding protein inhibitor (ID-1), E2F1, focal adhesion kinase (FAK), and HIF1α, thus promoting cardiac senescence. 46

| CIRCRNAS AND HCC
In accordance with the pivotal role in epigenetic and genetic regulation mentioned above, the deregulation of cir-cRNA expression has been reported in several diseases, such as cancers, neurological disorders, heart disease, diabetes, and atherosclerosis. 40,[47][48][49][50][51][52] In additional, the hypothesis of the intertwining of HCC with circRNAs is being validated.

| CircRNAs as reliable biomarkers for HCC
The early diagnosis of HCC in patients particularly is important. Due to their complex tissue-or cell-type and developmental stage-specific patterns, resistance to ribonucleases, such as exonucleases and RNase R, and longer half-lives, circRNAs are presumed to be ideal biomarkers. 15,16,18,19,[53][54][55] Based on microarray screening, our recent study identified 527 differentially expressed cir-cRNAs; hsa_circ_0005986, hsa_circ_0003570, and hsa_ circ_0004018 were significantly downregulated in HCC samples compared to adjacent noncancerous tissues and had the ability to differentiate HCC from liver cirrhosis and chronic hepatitis. [56][57][58] Other groups have found that circ-ZKSCAN1 is significantly downregulated in HCC samples compared with adjacent nontumorous tissues and is related to tumor number, cirrhosis, vascular invasion, and microscopic vascular invasion as well as tumor grade. 59  that hsa_circ_0007874 is downregulated in HCC and associated with poor patient survival. Hsa_circ_0001649 derived from the SHPRH gene is significantly downregulated in HCC samples and correlated with tumor size and the occurrence of tumor embolus. 62 Yu et al 63 revealed that the downregulation of hsa_circ_0001445 in HCC tissues is significantly correlated with aggressive characteristics and may serve as an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in HCC patients after hepatectomy.
Single-nucleotide polymorphisms (SNPs) may affect the susceptibility and clinical outcome of HCC. Guo et al 64 have found that rs10485505 and rs4911154 in circ-ITCH are significantly associated with increased HCC risk and can serve as susceptibility and prognostic biomarkers for HCC patients.
Exosomes may serve as good candidates for liquid biopsy, particularly for monitoring and predicting tumor occurrence and metastasis. 13,14 Li et al 50 found that circRNAs are abundant and stable in exosomes and that exosomal circRNAs might be a novel kind of promising biomarker for cancer diagnosis. The exosomal circRNAs associated with HCC need to be further deciphered.

| CircRNAs in HCC developmental mechanisms
As with other tumors, in HCC, an increasing number of studies has shown that circRNAs are good candidates for miRNA sequestration and the subsequent fine-tuning of target gene expression. 65,66 To date, most mechanistic studies of HCC have focused on miRNA sponges. Huang et al 65 revealed that hsa_circ_0000130 (hsa_circ_100338) functions as an endogenous sponge for miR-141-3p in the regulation of HCC invasion. Through a quantitative proteomics strategy, Yang et al 66 showed that epidermal growth factor receptor (EGFR), a validated miR-7 target, is present in CDR1asoverexpressing HepG2 cells. Our team has reported that hsa_circ_0005986 functions as an effective sponge for miR-129-5p, thereby regulating Notch1 expression. 58 Acting as a sponge for miR-9, hsa_circ_0007874 promotes p21 expression and then suppresses HCC progression, suggesting that hsa_circ_0007874 is a potential target for HCC treatment. 61 Based on RNA-sequence technology, Zheng et al 48 discovered that circHIPK3 (hsa_circ_0000284) is significantly upregulated in liver cancer compared with matched normal tissues and directly binds to miR-124, thereby upregulating Interleukin 6 receptor (IL6R) and Homeobox protein DLX2 expression. Chen et al 67 discovered that hsa_circ_0000284 promotes HCC proliferation and migration by directly binding to miR-124, thereby upregulating aquaporin 3 (AQP3) expression. By sponging miR-17-3p and miR-181b-5p, hsa_circ_0001445 promotes the expression of metalloproteinase inhibitor 3 (TIMP3) and then inhibits the proliferation and migration of HCC cells. 63 Zhu et al 68 demonstrated that hsa_circ_0067934 enhances the proliferation, migration and invasion of HCC cells via the sponging of miR-1324 and concomitant activation of the FZD5/Wnt/β-catenin signaling pathway. The validated circRNA-miRNA-target gene axis is shown in Figure 3. Characterized by mitochondrial dysfunction, growth arrest, and apoptosis, fatty liver disease is a high-risk factor for HCC. Gou et al 69 revealed that the circ_0046367-miR-34aperoxisome proliferator-activated receptor α axis underlies hepatic steatosis (Table 1).

| CircRNAs in HCC epithelialmesenchymal transition
The epithelial-mesenchymal transition (EMT) is defined as the process although which epithelial cells lose their differentiated properties and obtain mesenchymal characteristics. EMT plays important roles in HCC occurrence and progression. 70 EMT is regulated by complex transcriptional and posttranscriptional mechanisms. 71 CDR1as, a risk factor of hepatic microvascular invasion, is inversely correlated with the activities of miR-7. 22,23,66 MiR-7 downregulates the expression of EMT-associated signaling molecules, including EGF receptor (EGFR), insulin-like growth factor 1 (IGF1) and FAK, thereby indirectly upregulating E-cadherin and downregulating N-cadherin expression. 21 In view of the abovementioned miRNA sponge theory, we can deduce that CDR1as might be involved in the EMT process of HCC.

| CircRNAs as potential therapeutic targets in HCC
As mentioned above, circRNAs are intimately correlated with HCC carcinogenesis. What is the goal of circRNA application in HCC treatment? Hepatitis C virus (HCV) is an important latent carcinogenic factor for HCC. MiR-122 is a validated dispensable molecule for the HCV cell-life cycle. In recent times, Jost et al 72 successfully constructed artificial circRNAs containing an array of miRNA-122-binding sites, and these engineered circRNAs could not only sponge miRNA-122 and inhibit HCV replication but also affect HCV translation. Taken together, the involvement of circR-NAs and miRNAs in HCC provide experimental evidence that circRNAs may serve as novel therapeutic targets for HCC in the future.

| Prospect
An increasing number of circRNAs is involved in the deregulation of cancer pathophysiological processes, and these circRNAs have shown great potential in cancer diagnosis, prognosis, and therapy. 73 We can utilize plasmids to increase the expression of eukaryotic circRNAs and CRISPR/Cas9 or siRNA techniques to decrease circRNA expression. 74 The RT-droplet digital PCR (RT-ddPCR) method, a potent noninvasive and absolute quantification method, has been utilized to detect circRNA expression. 75 Moreover, to avoid tumor heterogeneous interference, we can use single-cell sequencing analysis to reveal the circRNA profile at the singlecell level. 76 The cancer-specific circRNA database (CSCD, http://gb.whu.edu.cn/CSCD) is a comprehensive database for exploring cancer-specific circRNAs. 77 CSCD will enable studies of the functions of cancer-associated circRNAs by predicting MRE sites, RBP sites and potential ORFs. At last, deregulated circRNAs not only facilitate the understanding of the complex etiology of HCC but also promise to be a kind of ideal biomarker.


extfieldsofstudy: Medicine Biology

text:
HOXA10-AS: A novel oncogenic long non-coding RNA in glioma

Glioma is the most common primary malignant tumor of the central nervous system. Emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) serve a major role of regulation in various types of human cancer, including glioma. However, the biological roles of thousands of lncRNAs remain unknown and require further identification. The present study investigated the functional role of lncRNA-HOXA10-AS in glioma. The present study examined the expression patterns of HOXA10-AS in glioma and normal brain tissues, as well as glioma cell lines and normal human astrocytes (HA) via reverse transcription-quantitative polymerase chain reaction. HOXA10-AS knockdown cells were generated using lentiviral short hairpin RNA against HOXA10-AS in A172 and U251 glioma cells. Cell growth was assessed by MTT assay, and a flow cytometer was used to investigate cell proliferation, cell cycle distribution and cell apoptosis. Western blot analysis was performed to analyze the expression levels of apoptosis-related proteins. HOXA10-AS was significantly upregulated in glioma tissues and cell lines, and increased HOXA10-AS expression levels were associated with higher grades of glioma. Knockdown of HOXA10-AS inhibited glioma cell proliferation and increased cell apoptosis rates compared with the control cells. HOXA10-AS markedly regulated the expression of the homeobox A10 (HOXA10) gene. Similarly, HOXA10 expression was increased with higher grades of glioma, and silencing of HOXA10 by small interfering RNA suppressed glioma cell proliferation and induced cell apoptosis. The results of the present study demonstrated that HOXA10-AS promoted cell growth and survival through activation of HOXA10 gene expression in glioma, which may potentially act as a novel biomarker and therapeutic target for clinical assay development.

Introduction
Glioma is the most frequent and aggressive primary malignant tumor of the central nervous system (CNS), and accounts for ~80% of primary CNS tumors. According to the 2016 World Health Organization (WHO) classification, gliomas are classified into four histopathological grades based on the degree of malignancy, including low-grade glioma (grade I and II) and high-grade glioma (grade III and IV), and glioblastoma (GBM) which is the highest grade of glioma (grade IV) (1). Despite significant improvements in the treatment of glioma over recent decades, including surgical resection, local radiotherapy and systemic chemotherapy, the prognosis of glioma patients remains poor, with a 5-year survival rate of 20-30% (2,3). Therefore, investigations into the molecular mechanisms and therapeutic targets of glioma have attracted extensive attention and have potential uses in the treatment of glioma (4,5).
With novel technologies having provided accelerating depths of RNA sequencing, recent studies have demonstrated that the human genome contains thousands of long non-coding RNAs (lncRNAs) (6). lncRNAs are >200 nucleotides in length and regulate gene expression by chromatin modification, at transcriptional and post-transcriptional levels (7,8). Functional studies for a number of lncRNAs have demonstrated that they participate in various aspects of cell biology, including cell proliferation, apoptosis, differentiation, invasion and metastasis, and potentially contribute to tumor initiation and malignant progression (9,10). lncRNAs have also been demonstrated to be involved in hematological malignancies (11) and multiple types of solid tumors, including hepatocellular carcinoma (12), breast (13), lung (14), gastric (15) and gynecological cancer (16), as well as melanoma (17). Downregulation or upregulation of lncRNA expression has been revealed to contribute to the transformed cancer phenotype, further affecting the clinicopathological appearance, prognosis and outcome of the cancer (18)(19)(20).

HOXA10-AS: A novel oncogenic long non-coding RNA in glioma
lncRNAs have been implicated in the oncogenesis of gliomas and are increasingly being considered as potential therapeutic targets. According to the function of lncRNAs in glioma, they are classified into oncogenes or tumor suppressors. HOTAIR (21,22), CRNDE (23), H19 (24,25) and XIST (26) are notable oncogenic lncRNAs, which can facilitate tumorigenesis. ADAMTS9-AS2 (27), CASC2 (28), MEG3 (29) and MALAT1 (30) are well-known tumor suppressor lncRNAs. Dysregulation of tumor suppressor lncRNAs leads to tumor formation. Therefore, advances in identifying glioma-related lncRNAs and elucidating their precise molecular mechanisms is critical for the diagnosis and treatment of glioma.
The present study identified a novel lncRNA, HOXA10-AS, which is transcribed from the antisense strand of the homeobox A10 (HOXA10) gene locus at chromosome 7p15.3. HOXA10-AS was markedly upregulated in glioma, and its increased expression levels were correlated with the malignancy of glioma. Furthermore, the present study addressed HOXA10-AS as a regulator of cell proliferation and apoptosis in glioma, and identified HOXA10 as a target of HOXA10-AS. Additionally, the oncogene HOXA10 had an important role in promoting the tumorigenesis of glioma.

Materials and methods
Patients and samples. A total of 59 glioma and 20 normal brain samples were obtained from the Department of Neurosurgery at the First Hospital of Jilin University (Changchu, China) from January to December of 2014. The 59 glioma patients (age range, 35-72 years; mean age, 46.6 years; 41 males and 28 females) consisted of 32 cases of low-grade glioma (WHO grade I and II) and 24 cases of high-grade glioma (WHO grade III and IV). All tissue samples were frozen in liquid nitrogen immediately after resection and stored in liquid nitrogen until use. All clinical pathological and biological data were available for these patients. The present study was approved by the Ethics Committee of The First Hospital of Jilin University and written informed consent was obtained from all patients. All the tumor tissues were obtained at primary resection, and none of the patients had undergone chemotherapy or radiation therapy prior to surgery.
Cell culture. The human glioma A172 and U251 cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and normal human astrocytes (HA) were obtained from ScienCell Research Laboratories (San Diego, CA, USA). A172 and U251 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), and HA cells were maintained in astrocyte medium (ScienCell Research Laboratories) at 37˚C in humidified atmosphere with 5% CO 2 .
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was isolated from tissues or cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA quantity was determined using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA synthesis was performed with 1 µg total RNA, using the PrimeScript™ RT reagent kit (Takara Biotechnology, Dalian, China), according to the manufacturer's protocol. Real-time PCR was performed on the Takara system, using the SYBR ® Premix Ex Taq™ II Kit (Takara Biotechnology). The cycling conditions were the following: 30 sec at 95˚C, followed by 40 cycles at 95˚C for 5 sec and 60˚C for 30 sec. GAPDH was used as the endogenous control. The relative expression was calculated using the 2 -ΔΔCq method (31). The primers of HOXA10-AS, HOXA10 and GAPDH are listed in Table I.
Lentiviral packaging and stable cell line establishment. A piLenti-shRNA-GFP lentiviral vector (Applied Biological Materials, Inc., Richmond, BC, Canada) was used for the HOXA10-AS-knockdown experiment. In brief, lentiviral particles expressing HOXA10-AS-specific or control scrambled shRNAs were co-transfected into 293T cells with the mixed set of packaging plasmids (SPAX2 and MD2G) using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The medium was changed after 8 h and the supernatant containing lentiviruses was collected 48 h later. Produced lentiviruses were concentrated using the Centricon Plus-20 centrifugal filter device (EMD Millipore, Billerica, MA, USA). Lentiviral stock was titred and stored at -80˚C. A172 and U251 cells were infected with the HOXA10-AS shRNA construct, and GFP + cells were sorted using a flow cytometer (FACSAria II; Becton-Dickinson, Mountain View, CA, USA). Subsequently, the cells were cultured with regular complete medium. Finally, the cells were analyzed for mRNA expression by RT-qPCR. The following two targets were used for HOXA10-AS knockdown: Target 1, ACA GGA AAC TAC CTA AAT CAC CGA CCA GT; and target 2, GTT CTG GTG CTG CCC GCG AAG GGC TGC CT.
MTT cell viability analysis. MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was performed to determine cell viability. A total of 1x10 3 cells were seeded into each well of 96-well plates and cultured in 200 µl growth medium. The cells were incubated with fresh complete medium every 2 days and after 6 days, 20 µg MTT was added to each well, followed by incubation at 37˚C for 4 h. The reaction was stopped by adding 150 µl dimethyl sulfoxide (DMSO) to each well and incubating at 37˚C for 10 min. The absorbance was then determined at 450 nm using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA).
BrdU cell proliferation assay. Cell proliferation was evaluated using BrdU Flow kit (BD Biosciences, San Diego, CA, USA). Cells were plated into 6-well plates with 2 ml complete growth medium/well, and prior to adding BrdU, the complete medium was replaced with serum-free medium. After 12 h, the serum-free medium was replaced with complete medium, and BrdU (1 mM/ml) was added to each well and incubated for 4 h. Subsequently, the cells were harvested, fixed and incubated with anti-BrdU and 7-AAD according to the manufacturer's protocol. Cell cycle distribution was determined using a flow cytometer (FACSAria II; BD Biosciences).
Cell apoptosis analysis. The Annexin V-PE assay kit (BD Biosciences) was used to analyze cell apoptosis in glioma cells. Cells were harvested at their exponential growth phase and were resuspended in 100 µl binding buffer at a density of 1x10 6 cells/ml. PE-conjugated Annexin V and 7-AAD reagent staining was performed at the concentrations and times recommended by the manufacturer. Stained cells were analyzed using a flow cytometer (FACSAria II; BD Biosciences), and data were analyzed using FlowJo V10 software (FlowJo LLC, Ashland, OR, USA).
Small interfering RNA transfection. HOXA10-knockdown in glioma cells was performed by siRNA transfection. siRNA oligonucleotides targeting HOXA10 were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), and the non-specific siRNA oligonucleotides (Shanghai GenePharma Co., Ltd.) were used as a negative control. Cells were transfected with 150 pmol siRNA/well in 6-well plates, using Lipofectamine RNAiMax Reagent (Invitrogen.), according to the manufacturer's protocol. All siRNA oligonucleotide sequences are listed in Table II.
Statistical analysis. All data are presented as the mean ± stan dard error of the mean (SEM) and were analyzed using Student's t-test. Spearman's correlation analysis was performed to investigate the correlation between HOXA10 and HOXA10-AS. All experiments were performed at least three times. All statistical analyses were performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

HOXA10-AS is overexpressed in human glioma samples and cell lines.
The human homeobox A (HOXA10) gene is located in the HOXA gene cluster at chromosome 7p15.3. A GeneBank search identified the gene HOXA10-AS in a tail-to-tail orientation relative to the HOXA gene, on the opposite strand of the HOXA10 gene (Fig. 1A). HOXA10-AS is a 1,159-bp long non-coding antisense transcript of HOXA10, consisting of three exons with a 3' polyadenylation tail (Fig. 1B).
To investigate the expression levels of HOXA10-AS in glioma, tumor tissues were collected from 59 patients with glioma and normal brain tissues were obtained from 20 healthy patients, and the expression levels of HOXA10-AS were examined using qPCR. The results demonstrated that HOXA10-AS was significantly upregulated in glioma tissues compared with expression in normal brain tissues (Fig. 1C). Furthermore, it was demonstrated HOXA10-AS expression was associated with the malignancy grade of brain tumors, and that HOXA10-AS expression in cases of high-grade glioma (WHO III and IV, n=27) was significantly higher than in cases of low-grade glioma (WHO I and II, n=32; Fig. 1D). Additionally, HOXA10-AS expression was analyzed in two human glioma cell lines, A172 and U251 cells, and normal human astrocytes (HA). Data demonstrated that HOXA10-AS expression was significantly increased in glioma cell lines compared with that in HA (Fig. 1E). Therefore, these results indicated that HOXA10-AS was frequently upregulated in glioma, and may serve an important role in the progression of malignant glioma.

HOXA10-AS promotes glioma cell proliferation.
To investigate the involvement of HOXA10-AS in glioma cell growth, the changes in cell viability and proliferation following silencing of HOXA10-AS were analyzed. The effects of  HOXA10-AS-knockdown were determined through lentivirus-mediated expression of shRNA targeting HOXA10-AS. These target sequences avoid overlap with the HOXA10 sequences, and do not cause silencing of the HOXA10 gene. The efficiency of HOXA10-AS downregulation was determined by RT-qPCR analysis. The results indicated that HOXA10-AS shRNA1 and shRNA2 induced significant reductions in the expression levels of HOXA10-AS, with a greater inhibitory effect with shRNA2 compared with shRNA1 in the two cell lines (Fig. 2A).
The growth inhibitory effects of HOXA10-AS-knockdown were confirmed by assessing the proliferation rate of cells for 5 consecutive days using MTT assays. In the scrambled control shRNA-infected cells, the number of cells increased by >10-fold over a 5-day culture period, whereas the number of HOXA10-AS-knockdown cells increased more slowly than that of the control cells. HOXA10-AS-knockdown cells cultured for 5 consecutive days exhibited statistically significantly lower proliferation rates than those of scrambled vector-containing cells ( Fig. 2B and C).
To illustrate the underlining mechanisms by which HOXA10-AS regulated cell growth, the cell cycle of A172 and U251 cell lines was then investigated using a BrdU array. HOXA10-AS-knockdown markedly decreased the proportion of S phase cells and increased the proportion of G0/G1 phase cells (Fig. 2D and E). HOXA10-AS-knockdown led to arrest in the growth of cells (A172 cells: 56.7%; U251 cells: 36.6%), compared with the control cells. Additionally, RT-qPCR demonstrated that the RNA expression levels of cyclin D1 and cyclin D2, cell-cycle regulators, were decreased in HOXA10-AS-knockdown glioma cells (Fig. 2F and G). These data indicated that knockdown of HOXA10-AS inhibited glioma cell proliferation via G0/G1 phase arrest and a decrease in DNA synthesis.
Therefore, HOXA10-AS functions by promoting tumor cell growth in glioma and may be a tumor oncogene.

Knockdown of HOXA10-AS induces glioma cell apoptosis.
In order to examine the effect of HOXA10-AS-knockdown on tumor cell apoptosis, cell apoptosis analysis was performed using flow cytometry in HOXA10-AS-knockdown and shRNA-control glioma cell lines. Knockdown of HOXA10-AS resulted in a marked increase in the proportion of Annexin V-positive and 7AAD-negative cells in knocked down A172 and U251 cells, compared with that of the scrambled shRNA-expressing cells (Fig. 3A and B). This result demonstrated that the k nockdown of HOXA10-AS induced glioma cell apoptosis.
In order to confirm the effect of HOXA10-AS on cell apoptosis, the protein expression levels of apoptotic regulators, including caspase, cleaved-caspase and Bcl-2 were investigated in HOXA10-AS-knockdown cells. Western blot analysis demonstrated that total caspase-3 and Bcl-2 protein expression levels were decreased and the protein expression level of cleaved-caspase-3 was increased following knockdown of HOXA10-AS in glioma cells (Fig. 3C and D). The expression trends of three apoptosis-related proteins were consistent with the change in the cell apoptosis rate following knockdown of HOXA10-AS.
These findings indicated that HOXA10-AS may serve a role in promoting glioma cell survival.
HOXA10-AS activates HOXA10 gene expression. In order to investigate the molecular mechanisms underlying the tumor promoting effects of HOXA10-AS on glioma cells, the ability of HOXA10-AS to regulate the transcription activity of the HOXA10 gene was investigated in A172 and U251 cells.
Sequence analysis demonstrated that exon 3 of HOXA10-AS overlaps with the second exon of HOXA10 by 607 bp in an antisense manner. The 607 bp of overlapping sequences is mostly located in 3'-UTR of HOXA10 mRNA, excluding 15 bp (Fig. 4A). Antisense lncRNAs have been implicated in the regulation of their overlapping sense transcripts (32,33). As a first step in determining whether HOXA10-AS regulates the expression of the HOXA10 gene, the present study analyzed the expression of HOXA10 in human glioma tissues and compared it with that in normal brain tissues. The results indicated that HOXA10 expression levels were significantly increased in glioma tissues (Fig. 4B). Additionally, HOXA10 mRNA expression levels in high-grade glioma tissues were higher than those in low-grade glioma tissues, and HOXA10 expression was associated with the degree of malignancy of the brain tumor (Fig. 4C). Further statistical analysis demonstrated a significant and positive correlation between HOXA10 and HOXA10-AS gene expression (Fig. 4D).
RT-qPCR analysis confirmed that HOXA10 expression was significantly decreased at the mRNA level in A172 and U251 cells transfected with shHOXA10-AS (Fig. 4E). These results indicated that HOXA10-AS regulated HOXA10 gene expression.

Knockdown of HOXA10 suppresses glioma cell proliferation and induces cell apoptosis.
To further examine the effect of HOXA10 on glioma cell growth, the present study analyzed the changes in cell proliferation and apoptosis following silencing of HOXA10 using transcript-specific siRNAs in A172 and U251 cells. These siRNA sequences avoid overlap with the sequences of HOXA10-AS, and do not cause gene silencing of HOXA10-AS.
The present study first assessed HOXA10 mRNA expression levels in A172 and U251 cells treated with siHOXA10 for 48 h. The results indicated that siHOXA10-2 and siHOXA10-3 induced a significant and visible decrease in HOXA10 expression levels, with a greater inhibitory effect achieved with siHOXA10-2 compared with siHOXA10-3 in the two cell lines (Fig. 5A and B). Following knockdown of HOXA10 in A172 cells, the percentage of cells in the S phase was decreased and the percentage of cells in the G0/G1 phase was increased (Fig. 5C), and caused a significant increase in the rate of cell apoptosis (Fig. 5D). Similar results were also obtained in glioma cell line U251 (Fig. 5E and F). These results indicated that knockdown of HOXA10 inhibited glioma cell growth by inducing G0/G1 phase arrest and cell apoptosis. Therefore, we hypothesized that HOXA10-AS was able to promote tumor cell growth by targeting HOXA10 in human glioma cells.

Discussion
It is currently well-known that >75% of the human genome is functional and encodes a large number of ncRNAs (34). Based on the ENCODE project, it is estimated that the human genome encodes >28,000 distinct lncRNAs, a number of which continue to be discovered and are yet to be given a functional annotation (35). Therefore, revealing the biological function of lncRNAs and identifying the disease-related lncRNAs are one of the most investigated areas in the life sciences and medical research fields.
To the best of our knowledge, the present study was the first to report the role of HOXA10-AS in glioma. The original results of qPCR analysis revealed significantly increased HOXA10-AS expression in glioma tissues and cell lines, and demonstrated that it was strongly associated with the histological tumor grades of glioma tissues. We hypothesized that HOXA10-AS functions as a tumor oncogene in glioma. In order to verify this hypothesis, the tumor oncogenic roles of HOXA10-AS were investigated in glioma cell lines. The present study investigated the function of HOXA10-AS in glioma by HOXA10-AS-knockdown. Compared with the control group, cell proliferation was significantly suppressed and the cell apoptosis rate was increased in shHOXA10-AS-transfected glioma cells. These data indicated that HOX10-AS served a crucial role in glioma tumorigenesis.
Certain lncRNAs are cis-acting, affecting the expression of neighboring genes. HOXA10-AS is a 1,161-bp spliced and polyadenylated RNA, which transcripts the antisense strand of the HOXA10 gene with a tail-to-tail overlap with HOXA10 transcript. Based on the results obtained in the clinical sample analysis, the expression of HOXA10 was upregulated in glioma and the expression of HOXA10-AS was correlated with that of HOXA10.
HOXA10, a member of the HOXA gene cluster, is a pivotal transcriptional regulator of early embryonic development. Previous studies have demonstrated that high expression of HOXA10 in several types of cancer is involved in regulating cell proliferation, migration and invasion, and is correlated with a poor prognosis (36)(37)(38)(39)(40). For example, HOXA10 significantly promoted nasopharyngeal carcinoma cell proliferation and invasion (36). Overexpression of HOXA10 predicted a poor prognosis in a subgroup of patients with epithelial ovarian cancer (37) or gastric cancer (38). HOXA10 serves a role in the migration processes of medulloblastoma cells (39).
Furthermore, Kurscheid et al (41) identified high expression of HOXA10 in glioblastoma tissues, compared with non-cancerous brain tissues based on Affymetrix array data from TCGA, and demonstrated that HOXA10 levels were significantly higher in glioblastoma stem cells. The present study revealed that HOXA10 exerted effects on promoting the proliferation and inducing the apoptosis of glioma cells. It was further verified that the expression of HOXA10 was decreased in HOXA10-AS-knockdown glioma cells compared with the control group, indicating that HOXA10 may be activated by high HOXA10-AS expression levels.
However, further studies are required to elucidate the detailed mechanism of how HOXA10-AS regulates HOXA10. The results of the present study demonstrated that HOXA10-AS is overlapped with the 3'-UTR of HOXA10 mRNA. Therefore, the potential HOXA10-AS/HOXA10 RNA hybrid may act to stabilize the HOXA10 transcript. It has been confirmed that Wrap53, as a natural p53 antisense transcript, is required for p53 stabilization via Wrap53/p53 RNA interaction (42). In addition, previous studies have reported that DNA methylation at key regulatory CpGs in the promoters of HOXA10 was significantly associated with HOXA10-signature expression (41). Antisense lncRNAs have been proposed to cause DNA methylation (43,44). Therefore, we hypothesized that the transcriptional activity of the HOXA10 gene may be regulated in part by HOXA10-AS-directed DNA hypomethylation. Several miRNAs were also known to regulate HOXA10 expression in colorectal cancer (45), oral cancer (46), hepatocellular carcinoma (47) and breast cancer (48), suggesting that HOXA10-AS may function as a competing endogenous RNA (ceRNA) to regulate HOXA10 gene expression.
In conclusion, the results of the present study provided the first evidence of HOXA10-AS values in glioma tissues, which is a tumor oncogene. Increased expression of HOXA10-AS was associated with malignancy status in glioma and knockdown of HOXA10-AS may be a potential therapeutic strategy for glioma. In vitro experiments demonstrated that silencing of HOXA10-AS suppressed glioma cell proliferation and induced cell apoptosis. Furthermore, the present study identified HOXA10 as a target of HOXA10-AS and demonstrated that knockdown of HOXA10-AS downregulated the mRNA expression levels of HOXA10. Additionally, HOXA10 exhibited definitive oncogenic effects in glioma. Further evaluation of the interactions between HOXA10-AS and HOXA10 is required. The results of the present study indicated an important role of HOXA10-AS in the promotion of the proliferation and survival of glioma cells and that it may serve as a novel biomarker and therapeutic target for glioma.
All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work were appropriately investigated and resolved.

Ethics approval and consent to participate
The present study was approved by the Ethics Committee of the First Hospital of Jilin University and written informed consent was obtained from all patients.