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Targeting myomiRs by tocotrienol-rich fraction to promote myoblast differentiation

Background Several muscle-specific microRNAs (myomiRs) are differentially expressed during cellular senescence. However, the role of dietary compounds on myomiRs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on myomiRs and myogenic genes during differentiation of human myoblasts. Young and senescent human skeletal muscle myoblasts (HSMM) were treated with 50 μg/mL TRF for 24 h before and after inducing differentiation. Results The fusion index and myotube surface area were higher (p < 0.05) on days 3 and 5 than that on day 1 of differentiation. Ageing reduced the differentiation rate, as observed by a decrease in both fusion index and myotube surface area in senescent cells (p < 0.05). Treatment with TRF significantly increased differentiation at days 1, 3 and 5 of young and senescent myoblasts. In senescent myoblasts, TRF increased the expression of miR-206 and miR-486 and decreased PTEN and PAX7 expression. However, the expression of IGF1R was upregulated during early differentiation and decreased at late differentiation when treated with TRF. In young myoblasts, TRF promoted differentiation by modulating the expression of miR-206, which resulted in the reduction of PAX7 expression and upregulation of IGF1R. Conclusion TRF can potentially promote myoblast differentiation by modulating the expression of myomiRs, which regulate the expression of myogenic genes.

Background
Satellite cells, which are located between the basal lamina and sarcolemma, act as vital components of the skeletal muscle tissue as they possess regeneration capacity. These satellite cells are mitotically quiescent and arrested at the G 0 phase. These cells express a limited number of genes and proteins [1]. In response to stress, such as muscle injury or physiological change, satellite cells are activated and undergo myogenesis, which involves a series of processes [2]. These cells migrate to the damaged site and withdraw from the G 0 phase to re-enter the cell cycle progression. The cells then undergo proliferation, differentiation and subsequently fuse with the adjacent muscle fibre to form a new muscle fibre [3]. In this proliferating state, the satellite cells are known as myoblast cells. Ageing gradually reduces the regenerative capacity of skeletal muscles resulting a decrease in the muscle mass and strength [4]. This contributes to age-or injury-induced muscle weakness leading to frailty in the elderly, which is one of the major health problems.
The myogenic program is controlled by various transcription factor families such as paired box gene family consisting of PAX3 and PAX7 and myogenic regulator family including MYOD1, MYOG, Myf5 and Myf6 [1,3]. The PAX7 transcription factor is required for muscle satellite cell biogenesis and specification of the myogenic precursor lineage [5]. Functioning upstream of the MYOD family, PAX7 is expressed in proliferating myoblasts, but is rapidly downregulated during differentiation. In mice, loss of PAX7 expression resulted in the differentiation of satellite cells into fibroblasts instead of myoblasts [6]. Most of the activated satellite cells proliferate, downregulate PAX7 and promote MYOD to progress into differentiation. Various growth factors and hormones such as insulin-like growth factor (IGF) [7], myostatin and follistatin [8], leukaemia inhibitory factors [9], hepatocyte growth factors and neuronal nitric oxide synthase are involved in muscle hypertrophy [10]. All of these modulators activate several pathways that modulate the expression of myogenic transcription factors.
MicroRNAs (miRNAs) have gained tremendous attention and provide a new avenue for understanding the regulatory mechanism of skeletal muscle development. miRNAs are evolutionary conserved small RNAs that have been identified as post-transcriptional regulators to suppress the expression of target genes. The suppression of gene expression is mediated by the binding of miRNA to the 3′ untranslated region (UTR) of the target mRNA [11]. miRNAs have been found to be involved in the regulation of various pathways that contribute to the modulation of several diseases, as a single miRNA can target several mRNAs. miRNAs are expressed in specific tissues, and those miRNAs that are specifically expressed in striated muscles are known as myomiRs [12]. Several myomiRs have been identified, including miR-1, miR-133a, miR-133b, miR-206, miR-486 and miR-499 [11]. Each myomiR has its own specific or overlapping target mRNA, which functions in promoting myoblast proliferation and differentiation and is differentially expressed during myogenesis. During the differentiation of myoblasts, the expression of miR-133b, miR-206 and miR-486 is elevated, resulting in the downregulation of PAX7 mRNA that promotes myogenic differentiation [5]. Thus, miRNAs play an irreplaceable role in the regulation of skeletal muscle differentiation.
Elucidation of the involvement of myomiRs during differentiation of human satellite cells provides current information of possible interactions between transcription factors, myomiRs and their target mRNAs, especially when modulated by dietary compounds. We proposed vitamin E as a promising agent to modulate the expression of myomiRs. Vitamin E consists of α-, β-, γand δ-tocopherol and α-, β-, γand δ-tocotrienol, all of which are potent lipid-soluble antioxidants. Vitamin E supplements have been found to prevent muscle damage [13]. However, the molecular mechanism of vitamin E in modulating muscle health remains elusive. Besides the tocopherol isomer, a mixture of tocotrienols particularly known as tocotrienol-rich fraction (TRF), which are less studied shows a better effect compared to the tocopherol single isomer [14]. TRF is commonly extracted from palm oil and consists of α-tocopherol and α-, β-, γand δ-tocotrienol. It has been reported to protect against oxidative damage and suppress reactive oxygen species (ROS) production [15]. A previous study showed that TRF prevents the replicative senescence of myoblast cells and promotes myogenic differentiation in which its activity is higher than the tocopherol isomer [16]. Interestingly, another study found that TRF prevents replicative senescence of fibroblasts by inhibiting the expression of miR-34a and increasing the expression of CDK4 [17]. As TRF is known to modulate miRNA expression, this study aimed to elucidate its modulatory role on myomiRs and myogenic genes during the differentiation of human myoblasts.

Materials and methods
Cell culture and serial passaging The Clonetics® Skeletal Muscle Myoblast Cell System containing normal human skeletal muscle myoblasts (HSMM; catalogue no. CC-2580, lot 0000257384), sourced from the quadriceps muscle of a 17-year-old female cadaver, was purchased from Lonza, USA. Cells were maintained in the growth medium, Skeletal Muscle Growth Media-2 (SkGM™-2 Medium) that consisted of SkBM™-2 Basal medium. The SkGM™-2 SingleQuots™ Kit [catalogue no. CC-3244 containing human epidermal growth factor (hEGF), dexamethasone, L-glutamine, foetal bovine serum (FBS) and gentamicin/amphotericin B (GA)]. Cell populations were trypsinised when they reached 70 to 80% confluency. For passaging, the culture medium was warmed to 37°C and the cells were seeded at 5000-7500 cells/cm 2 and were incubated at 37°C in a humid atmosphere containing 5% carbon dioxide (CO 2 ). At each passage, the number of divisions was calculated as log(N/n)/log 2, where N is the number of cells at the time of passage and n is the number of cells initially plated. The cells were divided into 2 groups, young cells with population doubling 14 (MPD 14) and senescent cells with population doubling 21 (MPD 21) [18].

Induction of differentiation
Differentiation medium was prepared by adding 2% horse serum to DMEM-F12 medium. To induce differentiation, both the groups of cells were plated at 20,000 cells/cm 2 in 24-well polystyrene cell culture plates (Thermo Fisher™ Nunc™, Waltham, USA) and incubated overnight in a growth medium in a cell culture incubator (37°C, 5% CO 2 ). The following morning, the growth medium was replaced with differentiation medium and for the TRF-treated groups, both young and senescent cells were treated with 50 μg/mL of TRF groups) [18]. The cultures were then incubated for 5 days.

Determination of myogenic purity
The myogenic purity of the cultures was monitored by determining the expression of desmin, a cytoskeletal protein that is expressed only in myogenic cells and not in fibroblasts. The number of desmin-positive cells, represented as a percentage of the total number of nuclei, was determined as the myogenic purity of the cell culture, and at least 500 cells were counted. Immunocytochemistry was performed using an antibody specific for desmin, at a dilution of 1:50 (clone D33; DAKO, Denmark). The cells were washed with × 1 phosphate-buffered saline (PBS) and fixed with 100% ethanol for 10 min. The fixation agent was removed by washing three times with × 1 PBS for 5 min. Non-specific binding sites were blocked with 1% FBS diluted in PBS for 30 min. The cells were then incubated with primary antibody against desmin. Specific antibody binding was detected using Alexa Fluor 488 (Invitrogen, USA) directly coupled to the secondary antibody at a dilution of 1:500. The nuclei were fluorescently detected by Hoechst staining (Sigma, USA) at a dilution of 0.0001% w/v. All images were digitalised using ImageJ software.

Quantification of the surface area of myotubes and myonuclei
Positive fluorescent areas of five randomly chosen fields from three individual experiments were evaluated. For each treatment, the mean area of the untreated group was used to calculate the percent increase or decrease in the area of myotubes.

Quantification of fusion index
To calculate the fusion index, the number of nuclei incorporated into the myotubes (> 2 nuclei) were counted and the ratio of this number to the total number of nuclei was determined.

TRF preparation and treatment
Gold Tri E 70 (Sime Darby Bioganic Sdn. Bhd., Malaysia) was used in this study. This Gold Tri E 70 consists of 25% α-tocopherol and 75% tocotrienol. Further, HPLC analysis of Gold Tri E 70 revealed that it consisted of 173.6 mg/g α-tocopherol, 193.4 mg/g α-tocotrienol, 26.2 mg/g β-tocotrienol, 227.7 mg/g γ-tocotrienol and 98.2 mg/mg δ-tocotrienol. TRF stock solution was freshly prepared in the dark by dissolving 1 g of Gold Tri E 70 (Sime Darby Bioganic Sdn. Bhd., Malaysia) in 1 mL of 100% ethanol (1:1) and stored at − 20°C for not more than 1 month. TRF was activated by incubating 45 μL of TRF stock solution (1 g/1 mL) with 60 μL of FBS, overnight at 37°C. To prepare TRF at a concentration of 50 μg/mL, 90 μL of DMEM with 10% FBS and 105 μL of 100% ethanol were added to the activated TRF, after which 600 μL of the mixture containing FBS and 100% ethanol (1:1) was added. The TRF solution (50 μg/mL) was prepared using the culture medium. Myoblasts were treated with 50 μg/mL TRF for 24 h, and untreated myoblasts were incubated with SKGM-2 medium (Lonza, USA) for proliferation analysis and with DMEM-F12 medium (Lonza, USA) for differentiation analysis. A series of dosage titrations performed in a previous study showed that 50 μg/mL of TRF treatment for 24 h produced the highest percentage of viable young and senescent myoblasts [16]. Furthermore, the myoblast cells used in the present study are similar to the ones in our previous study [16]. The media for both untreated and TRF-cells were changed simultaneously, and both groups of cells were harvested on the same day.

Primer design
Forward primers for microRNA were designed according to the miRNA sequences listed in the miRBase database (http://www.mirbase.org). For miR-486, miR-486-5p form was selected for forward primer synthesis. Table 1 shows the forward primer sequences for validated miR-NAs. Primers for human GAPDH, PAX7, IGF1R and PTEN were designed from listed NIH GenBank database using Primer 3 software and blasted with sequences in the GenBank database for specificity confirmation. The efficiency and specificity of each primer set was confirmed by melting profile evaluation. The primer sequences for quantitative gene expression analysis are shown in Table 2.

RNA extraction
Total RNA was extracted from different treatment groups of myoblasts using TRI Reagent (Molecular Research Center, Cincinnati, USA) according to the manufacturer's instructions. Polyacryl carrier (Molecular Research Center, Cincinnati, USA) was added to each extracted sample to precipitate the total RNA. Extracted RNA pellet was washed with 75% ethanol and dried prior to dissolving it in RNase-free distilled water. Aliquots of total RNA were stored at − 80°C immediately after extraction. The yield and purity of the extracted total RNA was determined by nanodrop spectrophotometer (Thermo Scientific, USA).

Real-time qRT-PCR
For quantitative analysis of miRNAs, reverse transcription (RT) was first performed with 10 ng of total RNA using Taqman microRNA Reverse Transcription kit (Applied Biosystems, USA) according to the manufacturer's instructions. PCR reactions were then performed to quantitate the expression levels of myomiRs (miR-206, miR-133b and miR-486) using Taqman Universal PCR Master Mix No AmpErase UNG (Applied Biosystems, USA), according to manufacturer's instructions, and Taqman microRNA assay kit (Applied Biosystems, USA) was used for the detection of myomiRs of interest. PCR amplification was performed in iQ5 Multicolor Real-Time PCR iCycler (Bio-Rad, USA) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. PCR reactions were performed in triplicates. The expression level of all myomiRs was normalised to the expression of RNU6B. The relative expression value (REV) of miRNAs was calculated using the equation for 2 −ΔCt method of relative quantification [16,19]: Expression of PAX7, IGF1R and PTEN genes was analysed using KAPA SYBR Fast One Step qRT-PCR kit (KAPA Biosystems, USA) and iQ5 Multicolor Real-Time PCR iCycler (Bio-Rad, USA). Each qRT-PCR mixture contained 11.7 μL nuclease-free water, 10 μL KAPA SYBR Fast master mix, 0.3 μL RT enzyme, 1 μL of 100 μM forward primer, 1 μL of 100 μM reverse primer and 1 μL total RNA (50-100 ng). Reactions were performed in iQ5 Multicolor Real-Time PCR iCycler (Bio-Rad, USA) at 42°C for 5 min and 95°C for 4 min, followed by 40 cycles of 95°C for 3 s and 60°C for 20 s. qRT-PCR reactions were performed in triplicates. GAPDH was used as a normalisation reference gene [19,20]. The relative expression value (REV) of the genes of interest was calculated using equation for the 2 −ΔCt method of relative quantification [16,19]:

Statistical analysis
Data are presented as the mean ± SD. Experiments were performed at least three times, and data were analysed by Student's t test and one-way analysis of variance (ANOVA). Significance was accepted at p < 0.05.

Results
Effects of TRF on the morphology and myogenic purity of skeletal muscle myoblasts Young myoblasts (PD 14) exhibited normal spindle shape with round nuclei (Fig. 1a, b, c), while senescent myoblasts were larger and flatter and consisted of prominent intermediate filaments (Fig. 1d, e). Senescent myoblasts exhibited different morphological features when treated with TRF. Most of the cells were spindle-shaped (Fig. 1f ), which resembled the TRF-treated young myoblasts (Fig. 1c). The myogenicity of the myoblasts was more than 90% in both treatment groups (Table 3). A comparison between different treatment groups showed that the myogenicity was similar in all treatment groups.

Effects of TRF on differentiation analysis of skeletal muscle myoblasts
The fusion index (Fig. 2) and myotube surface area (Fig. 3) were greater on days 3 and 5 than that on day 1 of differentiation. Ageing causes a significant reduction in the differentiation rate of senescent myoblasts on days 3 and 5 as compared to that of young myoblasts (control) (p < 0.05), as observed by decreased fusion index and surface area of myotubes. Treatment with TRF significantly increased the differentiation rate with an increase in the

TRF treatment modulates myomiR expression
Changes in the expression of miRNAs were observed in all groups of myoblasts. miR-133b expression was reduced significantly in senescent myoblasts during proliferation phase (Fig. 4a). However, there was a significant increase in the expression of miR-133b in young myoblasts when treated with TRF (p < 0.05). During the differentiation phase,  miR-133b expression was decreased in senescent myoblasts (p < 0.05). No significant change was observed in miR-133b expression when young and senescent myoblasts were treated with TRF during differentiation as compared to control group (Fig. 4b).
miR-206 expression was reduced significantly in senescent myoblasts during the proliferation phase (Fig. 4c). Upon TRF treatment, the expression of miR-206 increased significantly in young myoblasts (p < 0.05). During the differentiation phase, miR-206 expression in senescent myoblasts decreased significantly (p < 0.05). However, when treated with TRF, the expression of miR-206 increased significantly in young myoblasts from day 1 until day 3 of the differentiation phase and increased only on day 1 of differentiation phase in senescent myoblasts (Fig. 4d). Fig. 2 Effect of TRF on myoblasts differentiation. Fusion index was measured as an index of differentiation. a Denotes p < 0.05 compared to young control, b p < 0.05 compared to senescent control, c p < 0.05 compared to young treated, d p < 0.05 compared to day 1 of the same treatment and e p < 0.05 compared to day 3 of the same treatment. Data are presented as mean ± SD, n = 3 Fig. 3 Effect of TRF on myoblasts differentiation as measured by myotube surface area. a Denotes p < 0.05 compared to young control, b p < 0.05 compared to senescent control, c p < 0.05 compared to young treated, d p < 0.05 compared to day 1 of the same treatment and e p < 0.05 compared to day 3 of the same treatment. Data are presented as mean ± SD, n = 3 miR-486 expression was reduced significantly in senescent myoblasts during proliferation and differentiation phases (Fig. 4e, f ). However, upon TRF treatment, there was a significant increase in miR-486 expression in senescent myoblasts during the proliferation phase and on days 1 and 5 during the differentiation phase (p < 0.05).

TRF treatment modulates the expression of target genes and upstream regulators of myomiRs
Senescent myoblasts showed significantly decreased PAX7 expression during the proliferation phase (p < 0.05) (Fig. 5a). Upon TRF treatment, PAX7 expression was significantly increased in young myoblasts during proliferation. During the differentiation phase, there was a significant reduction in PAX7 expression in senescent myoblasts (p < 0.05). Treatment with TRF during the differentiation phase caused a significant decrease in PAX7 expression in young myoblasts on days 3 and 5 and on days 1 and 5 in senescent myoblasts (Fig. 5b).
PTEN expression increased significantly in senescent myoblasts during proliferation and differentiation phases (p < 0.05) (Fig. 5c, d). TRF treatment caused a significant reduction in PTEN expression in young and senescent myoblasts during the proliferation phase. During the  -133b (a, b), miR-206 (c, d) and miR-486 (e, f) in young control myoblasts, TRF-treated young myoblasts, senescent control myoblasts and TRFtreated senescent myoblasts. a Denotes p < 0.05 compared to young control, b p < 0.05 compared to senescent control and c p < 0.05 compared to young TRF-treated myoblasts. Data are presented as relative expression value (REV) normalised to RNU6B expression (mean ± SD, n = 3) differentiation phase, treatment with TRF decreased PTEN expression in senescent myoblasts on day 1 of differentiation (Fig. 5d).
Senescent myoblasts exhibited significantly decreased IGF1R expression during the proliferation phase and on day 3 of the differentiation phase (p < 0.05) (Fig. 5e, f ). TRF treatment caused a significant increase in IGF1R expression in young and senescent myoblasts during the proliferation phase. During the differentiation phase, treatment with TRF caused a significant increase in IGF1R expression in senescent myoblasts on day 1, which decreased on day 5 (Fig. 5f).

Effects of TRF on cell cycle profile
Analysis of cell cycle profile at day 0 showed that myoblast population in the G 0 /G 1 phase was significantly higher and in the S phase, the population of senescent cells was significantly lower than those in the young cells (p < 0.05) (Fig. 6). Treatment with TRF caused a significant reduction in the percentage of senescent myoblasts in the G 0 /G 1 phase and a significant increase in the percentage of young and senescent myoblasts in the S phase (p < 0.05) (Fig. 6e).
On day 1 of differentiation, the percentage of myoblasts in the G 0 /G 1 phase was significantly higher in  PAX7 (a, b), PTEN (c, d) and IGF1R (e, f) in young control myoblasts, TRFtreated young myoblasts, senescent control myoblasts and TRF-treated senescent myoblasts. a Denotes p < 0.05 compared to young control, b p < 0.05 compared to senescent control and c p < 0.05 compared to young treated myoblasts. Data are presented as relative expression value (REV) normalised to GAPDH expression (mean ± SD, n = 3) senescent cells than that in the young cells (p < 0.05), while the percentage of cells in the S and G 2 /M phases was significantly reduced in both groups of myoblasts (Fig. 7). A comparison of the cell cycle profile between days 0 and 1 of differentiation showed a significant difference in the percentage of cells in the G 0 /G 1 , S and G 2 /M phases in both groups (p < 0.05).

Discussion
The integrity of myoblast cell structure as well as the formation of myotubes is maintained by the cytoskeleton, cell membrane and the extracellular matrix (ECM) [21]. Young myoblasts are morphologically spindle-shaped and elongated structures. In contrast, senescent myoblasts manifest morphological changes with a flattened structure and larger cytoplasm. Upon induction of differentiation, myoblasts fuse together to form a multinuclear myotube. Young myoblasts form large-branched myotubes, whereas senescent myoblasts form smaller myotubes [16]. As cells senesce, the level of reactive oxygen species (ROS) increases proportionately. In addition, the level of antioxidants is inversely proportional to the level of ROS throughout the senescence process. Accumulation of ROS in the cells induces oxidative stress, which causes oxidative damage to macromolecules such as DNA, RNA, protein and lipid [22]. Consequently, several pathways and cellular metabolism are altered, which leads to changes in the cytoskeleton, cell membrane and the ECM [23], resulting in phenotypic changes in senescent myoblasts, as observed in the present study. Therefore, introducing antioxidants to the altered system is predicted to reduce the oxidative stress, thus delaying the senescence of myoblasts.
Vitamin E, particularly TRF, plays a pivotal role in scavenging peroxyl radicals and prevents the peroxidation of macromolecules, thus improving the oxidative status of cells [24]. Vitamin E consists of two isomers, tocopherol and tocotrienol. Tocotrienol has been reported to possess better antioxidant activity and effectively reduces the oxidative stress in lipophilic environment [25]. In this study, TRF treatment ameliorated the morphological structure of senescent myoblasts and showed similar features to young myoblasts. A previous study showed that TRF treatment of senescent fibroblast cells reversed the morphological structure to form young fibroblasts [26]. Similarly, in a previous study by Khor et al., it was reported that senescent myoblasts treated with TRF appeared to have similar morphological features as young myoblast cells [16]. This observation could be due to the modulation of protein expression, which is involved in maintaining cell structure. Matrix metalloproteinase (MMP), responsible for the degradation of procollagen, is highly expressed in senescent cells. This protein alters cell structure maintenance in senescent cells [27]. However, TRF increases the expression of procollagen in senescent fibroblast cells [28], hence improving the morphological structure of senescent cells as observed in this study.
Homeostasis between proliferation and differentiation of myoblast cells during myogenesis is tightly regulated to prevent uncontrolled proliferation [29]. In the present study, the percentage of senescent myoblast cell population was higher in the G 0 /G 1 phase and lower in the S Fig. 7 Cell cycle profile of young and senescent myoblasts at day 1 of differentiation. Flow cytometry analysis of cell cycle progression of young control myoblasts (a), TRF-treated young myoblasts (b), senescent control myoblasts (c) and TRF-treated senescent myoblasts (d). Quantitative analysis of cell cycle progression of young and senescent myoblasts (e). a Denotes p < 0.05 compared to young control, and d p < 0.05 compared to same treatment at day 0 of differentiation. Data are expressed as mean ± SD (n = 6) phase than young cells, during proliferation. A similar result was also observed in differentiated senescent myoblasts. Interestingly, TRF treatment of both proliferating young and senescent myoblast cells enhanced the cell cycle progression as the cell population in the G 0 /G 1 and S phases reduced and increased, respectively. However, during the induction of differentiation, TRF promotes cell cycle withdrawal in young myoblast cells. Like other somatic cells, the proliferation capacity of myoblast cells is limited by replicative senescence due to progressive loss of telomere length [30]. In order to prevent tumour progression, cell cycle checkpoints act as barriers to prevent the replication of damaged DNA whereby cells are arrested at the G 0 /G 1 phase [29].
All cell cycle checkpoints are regulated by several cyclin-dependent kinases (CDKs) and cyclin proteins. Depending on the stimuli or cell environment such as DNA damage response (telomere shortening), CDK inhibitors such as p16 or p21 are expressed to inhibit the formation of CDK/cyclin complex, thus arresting cell cycle progression at the G 0 /G 1 phase [31]. Previous studies have shown that TRF treatment of senescent fibroblast cells increased the expression of telomerase and enhanced the elongation of telomere [26]. Furthermore, γ-tocotrienol treatment of senescent cells downregulates p16, cyclin D1 and hypophosphorylated-Rb, all of which are involved in cell cycle arrest [32]. Thus, TRF is postulated to modulate telomerase expression, increase the expression of proteins involved in cell cycle to prevent cell cycle arrest and promote proliferation of myoblasts. Treatment with TRF increased the percentage of myoblast cells in the G 0 /G 1 phase on day 1 of differentiation induction indicating the promotion of cell cycle arrest and inhibition of cell proliferation for differentiation to occur. This could be due to TRF, which is dependent on cell environment or stimuli. Previous studies have shown that γand δ-tocotrienol stimulates the differentiation of osteoblasts, which in turn enhance bone formation [33]. Furthermore, a previous study has shown that combined activity of TRF is much better in promoting myoblast differentiation than single α-tocotrienol treatment [16]. However, complete understanding of the mechanism of TRF in promoting the proliferation and differentiation of myoblasts remains elusive.
At the molecular level, regulation of proliferation and differentiation of myoblast cells during myogenesis is associated with several genes and myomiRs (miR) [11]. During cell renewal, quiescent satellite cells upregulate the expression of PAX7 and downregulate the expression of its myogenic regulatory factor (MRF) gene targets, MYOD1 and MYOG. Expression of PAX7 promotes re-entry of quiescent satellite cells into cell cycle progression and enhances the proliferation of myoblasts [6]. In the present study, the expression of PAX7 gene was increased in differentiated myoblasts, and this increased expression remained constant after several days of differentiation induction. However, TRF treatment increased the expression of PAX7 gene during proliferation and downregulated its expression during differentiation. Increased expression of PAX7 followed by suppression of myogenesis inhibitors Id2 and Id3 has been reported to upregulate the expression of MYOD1 and MYOG [34]. MYOD1 is directly involved with the activation of p21, cyclin D3 and Rb expression, which are critical for irreversible cell cycle withdrawal of myoblast cells from the G 0 /G 1 phase during differentiation and terminal differentiation phases [35].
PAX7 expression is regulated by miR-206 and miR-486. As MYOD1 expression is increased, this transcription factor that has its binding site in the promoter regions of miR-206 and miR-486, facilitates the expression of these two myomiRs [5]. Interestingly, TRF treatment upregulated the expression of miR-206 during proliferation, and this expression was further upregulated during differentiation. Another myomiR, miR-486, was upregulated when treated with TRF in proliferated and differentiated myoblasts. In contrast, TRF treatment did not upregulate the expression of miR-486 during differentiation. Previous studies have shown that the suppression of the PAX7 gene by miR-206 and miR-486 enhanced the commitment of myoblast cells to differentiate [5]. However, overexpression of PAX7 promotes uncontrolled proliferation [36]. Therefore, in the present study, TRF might play a role in maintaining the proliferation and differentiation of myoblasts by modulating the expression of PAX7 gene, miR-206 and miR-486, without disturbing the homeostasis of myogenesis.
Various modulators that regulate the activity of satellite cells and utilise various signalling pathways, including the IGF1R/P13K pathway, control myogenesis. This pathway mediates the functions of IGF as both IGF-1 and IGF-2 bind to IGF1R. IGF1R was downregulated during the late differentiation stage due to the presence of miR-133 response element (MRE) located in the 3′ UTR [9,37]. This would explain the direct effects of miR-133 towards IGF1R as a negative regulator of PI3K/ Akt. miR-133 downregulates Akt phosphorylation via inhibition of IGF1R protein, which is responsible for glucose metabolism, cell proliferation and apoptosis [37,38]. A reduction in Akt phosphorylation was observed during the differentiation of C2C12 myoblasts. miR-133 is important to regulate and balance the activity of IGF in muscle cells. IGF1R was found to be deregulated in rhabdomyosarcoma (RMS) where its expression increased consistently and, hence, is suggested as an initial factor responsible for oncogenic transformation of muscle cells [39]. Prolonged and consistent IGF1R expression resulted in increased proliferation and prevention of the differentiation phase [37].
Akt activation also activates mTOR and inhibits GSK3B, a negative regulator of protein synthesis and muscle growth. PTEN is a PI3K phosphatase that deactivates Akt, inhibiting muscle cell growth and muscle cell survival [40]. Decreased PTEN expression stimulates the PI3K/Akt pathway for the promotion and expression of myogenic transcription factors such as MYOD1, MYOG and Myf5 during myoblast proliferation and differentiation. A previous study reported reduced expression of miR-486 in Duchenne muscular dystrophy [41] and ageing [42]. miR-486 acts as a mediator for MYOD1 and regulates the PI3K/Akt pathway. miR-486 is transcribed from an intron of the Ank1 gene consisting of 39a exon code for muscle-specific Ank1 protein, which connects the sarcomere to the sarcoplasmic reticulum [43]. The expression of Ank1 gene transcript is controlled by a promoter site that contains two conserved E-boxes, which interact with MYOD [43]. An increase in the expression of miR-486 by TRF shows that TRF possesses the ability to delay the ageing phenotype and sarcopenia during ageing.
In the present study, TRF treatment has also been shown to increase the expression of myomiRs in young and senescent myoblasts in both proliferation and differentiation phases. Thus, TRF might play a role in the biogenesis of myomiRs directly or indirectly, which involves several processes [11]. Initially, the myomiR is transcribed in the nucleus as a primary transcript or pri-myomiR with a stem-loop structure. Here, the transcription process is modulated by various transcription factors. The pri-myomiR is subsequently processed to form pre-myomiR by the removal of both the end strands. Later, in the cytoplasm, the pre-myomiR loop is cleaved out and unwound by a helicase to form a single-stranded mature myomiR. These modification processes involve various proteins and may be one of the direct or indirect targets of TRF. At the transcriptional level, miR-206 is regulated by transcription factor FOXO3a [11]. A previous study showed that TRF treatment increases the expression of FOXO3a gene [44]. Another finding also reported that γand δ-tocotrienol increased the expression of FOXO3a gene [45]. Therefore, TRF is predicted to modulate the biogenesis of myomiR via regulation of its transcription factor.
As there was a decrease in the expression of myomiRs in senescent myoblasts, we proposed another mechanism for TRF-mediated regulation of myomiRs, which may be attributed to its radical-scavenging effect. The RNase III enzyme, Dicer, is responsible for cleaving the loop out of pre-miRNA (a major steps in the biogenesis process) to produce double-stranded mature miRNAs [11]. This enzyme is inhibited by various stress factors including ROS, which is accumulated during ageing [46]. Another finding showed that Dicer expression decreased with increased level of oxidative stress and DNA damage. As TRF effectively reduced the levels of ROS, especially in senescent cells, TRF is suggested to modulate myomiRs by reducing the oxidative stress, which in turn enhances the activity and expression of Dicer. Therefore, TRF may be involved in the biogenesis of myomiRs via modulation of Dicer expression. Hence, to verify the specificity of TRF response on Dicer expression, a further study is required by using other antioxidants or by inhibiting the function of miRNA by anti-miR oligonucleotides.
TRF naturally exists as a mixture of various forms of vitamin E; all tocotrienol forms are present and highly concentrated in TRF. However, each cell has its own preference for various forms of vitamin E. A previous study showed that the concentration of αand δ-tocotrienol was the highest in myoblasts, [18] while γand δ-tocotrienol were the most abundant forms in fibroblasts [47]. As previously described, TRF treatment showed a better effect than single isomer treatment. Hence, preferential and selective uptake of vitamin E form by the cell represents the best synergistic effect between vitamin E forms and suitability, which depends on cell environment. Figure 8 summarises the modulatory effect of TRF on the expression of myomiRs and the myogenic regulatory factors. Our results revealed that TRF is a potential muscle differentiation agent that modulates the expression of myomiRs and its target genes involved in myoblast differentiation during myogenesis.

Conclusion
The findings of the present study demonstrated that tocotrienol-rich fraction with antioxidant and non-antioxidant properties altered the expression of myomiRs, specifically miR-133b, miR-206 and miR-486, thereby modifying the expression of their target genes that are involved in myogenesis to promote muscle differentiation in young and senescent myoblasts.


extfieldsofstudy: Medicine Biology

text:
Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

ABSTRACT A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses. IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

S uccessful elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope (Env) protein through vaccination remains an important but unachieved goal. It is known that 15 to 20% of individuals chronically infected with HIV-1 are capable of eliciting bNAbs (1)(2)(3)(4). These individuals first develop NAbs (5,6), which drive viral escape and evolution, resulting in expansion of Env diversity (4,(7)(8)(9). In some patients, this sequence diversity drives the development of bNAbs capable of targeting conserved epitopes (10)(11)(12)(13). These studies suggest that the long-term exposure of the immune system to multiple diverse Env sequences can result in the development of bNAbs.
No HIV-1 vaccine to date has been capable of eliciting bNAbs in humans (14)(15)(16)(17). A variety of strategies have been explored with the goal of expanding the breadth of vaccine-elicited NAbs. One strategy assessed mixtures of different Envs with the goal of exposing B cells to sequence diversity, but this approach did not appreciably improve the breadth of tier 2 NAb responses (18)(19)(20)(21)(22)(23). Additionally, groups have utilized rationally designed immunogens focused on eliciting bNAbs to a single Env epitope, such as the CD4 binding site, but these immunogens have not driven the full development of such bNAbs (24)(25)(26). Mimics of the native HIV-1 Env trimer, such as the SOSIP trimer, have also been assessed, but they elicited NAbs with minimal breadth and targeted a hole in the glycan shield (27)(28)(29)(30). Finally, long-term vaccination strategies have been considered, with the goal of allowing for affinity maturation and the development of neutralization breadth. A few studies have explored vaccination regimens spanning multiple years; however, they have also failed to induce broad tier 2 neutralization (31).
In this study, we evaluated the effects of a longitudinal prime/boost vaccination regimen on the evolution of binding and NAb responses in guinea pigs over a vaccination regimen that spanned more than 2 years. We found that multivalent, sequential prime/boost vaccination regimens improved the breadth of binding antibodies compared to vaccination with a single Env. Additionally, while we observed a limited breadth of tier 2 NAbs in all vaccination regimens, the breadth and magnitude of these NAbs did not increase over the course of the longitudinal regimen. These data suggest that novel immunogen design strategies and vaccination regimens will be needed to improve tier 2 NAb responses.
Binding antibody responses. We first assessed the ability of each vaccination regimen to elicit binding antibodies to a multiclade panel of vaccine-matched gp140 proteins by enzyme-linked immunosorbent assay (ELISA) (Fig. 1B). All guinea pigs elicited comparable and robust binding antibody responses after the second vaccination. The highest titers were seen at weeks 16, 78, and 120, which correspond to peak immunogenicity, and there were contractions in the overall Env-specific binding antibody responses after long-term rests (weeks 62, 104, 138, and 200). For all groups, the IgG1 responses were the highest, followed by the IgG2, IgA, and IgM responses (Fig.  1C). Antibody avidity in all groups increased similarly over time, peaking at week 78, where it plateaued until week 200 (Fig. 1D). Mucosal IgG responses were also detected in the majority of animals, but at lower titers (data not shown).
Mapping binding antibody responses. We mapped binding antibody responses by competition ELISAs using the following bNAbs: 3BNC117 to the CD4 binding site (CD4bs) (35), PG9 to variable loop 2 (V2)/glycans (36), PGT121 to V3/glycans (37), and 447-52D to V3 (38). Sera from all groups outcompeted 3BNC117 binding to C97 gp140 similarly, suggesting that all vaccine regimens elicited binding antibodies in the vicinity of the CD4bs (Fig. 2A). The ABCM and 4C regimens were able to outcompete PG9 binding more successfully than the C97 regimen, with this effect increasing from week 16 to 78 (Fig. 2B). The 4C regimen was superior to the C97 regimen at outcompeting PGT121 and 447-52D binding ( Fig. 2C and D). These data suggest that while the binding antibodies target multiple regions of Env, the 4C regimen induced the most robust V3-directed binding antibodies.
Assessing the magnitude of binding antibodies to linear peptides across the HIV-1 envelope. We next mapped linear epitope binding antibodies to HIV-1 gp140 elicited by each vaccination regimen by utilizing a peptide microarray (39). The majority of early responses were elicited to V3 at week 16, although responses to V2 were boosted over the course of the vaccination regimen and persisted to week 200 (Fig. 3A). Interestingly, there were large increases in the magnitude of linear binding responses for both V2 and V3 from week 104 to week 120 that gradually declined until week 200. We also found that all vaccination regimens also elicited robust V1/V2 binding antibodies using V1/V2 scaffolds (Fig. 3B) (40,41). Furthermore, at weeks 78 and 120, the 4C regimen elicited binding antibodies to a statistically greater number of V2 peptides than C97 alone (Mann-Whitney U test, P Ͻ 0.05). Similarly, the ABCM vaccine elicited Guinea pig polyclonal antibody avidity as measured by urea disruption ELISA. Each dot represents the result for an individual animal, and error bars represent the standard deviations. Percent avidity was calculated using the following formula: [(absorbance of urea-treated sample/absorbance of non-urea-treated matched sample) ϫ 100]. Zero to 30% is low avidity, 30 to 50% is moderate avidity, and Ͼ50% is high avidity. The 80% bar is used as a reference point within the high-avidity region.
binding antibodies to a statistically superior number of V2 peptides at week 120 than C97 alone (Mann-Whitney U test, P Ͻ 0.05) (Fig. 3C). All vaccines elicited binding antibodies to similar regions of V2 across all time points, with the multivalent vaccines eliciting antibodies to greater numbers of peptides within these regions at weeks 78, 120, and 138 (Fig. 3D). Additionally, the multivalent vaccines elicited binding antibodies capable of binding to greater numbers of sequences within different clades and circulating recombinant forms (CRFs) than did C97 vaccines (Mann-Whitney U test, P Ͻ 0.05) (Fig. 3E).
Tier 1 neutralizing antibody responses. We next assessed the neutralization capacity of the antibodies utilizing a multiclade tier 1 panel of neutralization-sensitive pseudoviruses using the TZM.bl neutralization assay (42,43). Against specific tier 1 pseudoviruses (SF162.LS, BaL.26, SS1196.1, and 6535.3), the 4C and ABCM regimens generally elicited higher NAb titers than did the C97 regimen (Mann-Whitney U test, P Ͻ 0.05) (Fig. 4A). For one pseudovirus, Q23.17, no neutralization was observed at week 16 but clear neutralization emerged by week 120, indicating that repetitive boosts over a prolonged period of time can increase the breadth of tier 1 NAbs. However, the NAb titers against most tier 1 pseudoviruses assessed did not increase over time.  Tier 2 neutralizing antibody responses. We next assessed NAb responses to tier 2 viruses, which are representative of circulating strains of HIV-1 and are more neutralization resistant than tier 1 viruses. For these studies, we utilized a defined, global, tier 2 panel of pseudoviruses selected to represent global HIV-1 sequence diversity (44).  Guinea pig sera obtained prevaccination (week 0) and at weeks 16, 62, 78, 104, and 120 were tested against a multi-clade panel of tier 1 isolates in the TZM.bl neutralization assay. Groups were vaccinated with the ABCM (n ϭ 4), 4C (n ϭ 5), or C97 (n ϭ 5) regimen. The test pseudovirus, its tier, and clade are shown above each panel. The MuLV background signal for each animal-matched MuLV control was subtracted from neutralization data for all data points. Error bars show the standard deviation for each group at each time point. The limit of detection for the assay is a 50% infective dose (ID 50 ) titer of 20. Asterisks denote statistically significant differences from the results for C97 vaccination alone (P Ͻ 0.05, Mann-Whitney U test). (B) Magnitudes of antibody-dependent neutrophil phagocytosis (ADNP) for guinea pig samples. Guinea pig sera obtained prevaccination (naive) and at week 120 were tested in an ADNP assay as described in Materials and Methods. Groups were vaccinated with the ABCM (n ϭ 4), 4C (n ϭ 5), or C97 (n ϭ 5) regimen. Each dot represents a sample tested in triplicate, and error bars show standard deviations for each vaccination group. Controls included human HIV-positive serum, human HIV-negative serum, and no-antibody samples. Phagocytic score was determined using the following formula: (% bead-positive SSC high , CD11R1 ϩ , and CD4 Ϫ cells ϫ MFI of bead-positive cells)/10,000.
Purified IgG was used to reduce the low nonspecific background signal in the assay. We observed positive NAb responses to tier 2 pseudoviruses X1632 (clade G) and 25710 (clade C) for the week 120 samples, with the highest-magnitude responses in the 4C group (Student's t test, P Ͻ 0.05 for X1632) (Fig. 5A). We evaluated the kinetics of the modest tier 2 responses over time and found that these responses were initially raised at week 16 for all vaccines tested (Fig. 5B). By week 78, the 4C regimen elicited higher NAb responses than the ABCM group against X1632, and this result was maintained for the duration of the entire study (Student's t test, P Ͻ 0.05). The 4C regimen also elicited higher NAb responses than the ABCM group against 25710 at week 78. Additionally, the 4C group elicited higher NAb titers than the C97 group against X1632 at weeks 120 and 200 (Student's t test, P Ͻ 0.05) (Fig. 5B). The tier 2 responses did not increase in magnitude or breadth over the course of the vaccination regimen. Thus, all vaccination regimens elicited limited and modest tier 2 NAbs, particularly the 4C regimen, but repetitive boosting over 2 years was unable to expand these tier 2 NAbs.
Mapping the specificities of tier 2 neutralizing antibody responses. Finally, we conducted functional mapping studies to determine the targets of the tier 2 NAbs. Both the 4C and C97 regimens elicited detectable neutralization titers to tier 2 virus JRCSF (Fig.  6A). Animals that elicited the highest NAb responses to JRCSF were utilized for mapping studies (guinea pigs 278, 279, 281, and 283). This tier 2 neutralization could not be . Horizontal black lines indicate mean titers, and gray dots indicate responses below the limit of detection of the assay. Red, blue, and black dots represent responses that were both detectable and greater than the value for the MuLV control. Each dot represents the result for a single guinea pig, colored according to vaccination regimen. In the C97 vaccination group, one guinea pig died at week 134 and one at week 177 due to age. Horizontal brackets denote statistically significantly different results (P Ͻ 0.05, Student's t test). outcompeted by RSC3 (CD4bs target) (Fig. 6B) or by using JRCSF with an S334A mutation (encoding a change from S to A at position 334; V3 target) (Fig. 6C), suggesting minimal CD4bs or V3/glycan-directed neutralization. In contrast, the neutralization of JRCSF was largely but not completely competed by a V3 linear peptide from Bal (Fig. 6D).

DISCUSSION
In this study, we evaluated the immunogenicity of repeated boosting with homologous and heterologous Env gp140 vaccination regimens over the course of more than 2 years in guinea pigs. The multivalent 4C and ABCM regimens elicited greater breadths of V2binding antibodies than the monovalent C97 regimen. Limited and modest heterologous tier 2 NAbs were also induced, particularly by the 4C regimen, with responses largely directed against a linear epitope in V3. Tier 2 NAbs did not increase in magnitude or breadth following repeated boosting over the 120-week vaccination regimen. These data suggest that novel immunogen design and vaccination strategies will likely be needed to induce broad, high-titer tier 2 NAbs.
Few HIV-1 vaccination studies have explored vaccination regimens spanning more than 2 years in duration. Saunders et al. conducted a study over a 4-year period which was capable of eliciting glycan-dependent, V3-directed NAbs after repeated vaccination with a single Env sequence in rhesus monkeys (31). These monkeys elicited tier 2 NAbs to kifunensine-treated pseudoviruses, but not to viruses containing wild-type glycosylation patterns. Our study, in contrast, utilized both homologous and heterologous prime/boost regimens in guinea pigs to assess the role that multivalency may play in improving NAb responses, and the 4C vaccine improved the achieved neutralization magnitude over the course of a long-term vaccination regimen. Animals elicited V3-directed NAbs able to neutralize wild-type tier 2 pseudoviruses, and neutralization was not dependent on the S334 glycan. Our study, however, was limited, as four Env sequences do not represent the total sequence diversity of viruses that would be found in an HIV-1-infected individual capable of eliciting bNAbs, and increased sequence diversity may be required to drive bNAb breadth (45,46).
Interestingly, over the course of the longitudinal vaccination regimen, antibodies to linear epitopes and tier 1 NAbs evolved, while the tier 2 NAb responses remained largely unchanged over time. While there were increases in the breadth of V2-binding antibodies and tier 1 NAbs, this did not result in an increased magnitude or breadth of tier 2 NAbs. These data suggest that a longitudinal vaccination alone will not serve to drive tier 2 neutralization breadth. The limited tier 2 NAb responses induced in this study were largely directed to a linear epitope within the V3 loop. This is not surprising, given that V3 is an immunodominant epitope within the Env protein (47) and that gp140 immunogens sample various conformations, some of which expose V3 (48,49). Monoclonal antibodies that target linear epitopes in V3 against a limited breadth of tier 2 viruses have previously been characterized, including 447-52D and 3074 (38,50). Similar V3-directed NAbs have also been isolated from HIV-1-infected individuals and have been elicited in a number of vaccine studies (27,28,(51)(52)(53). Utilizing strategies to reduce V3 exposure and immunogenicity may assist in increasing the breadth of tier 2 NAb responses. Studies with native-like SOSIP trimers, however, have to date not been able to expand the breadth of tier 2 NAb responses, and thus, utilizing a native-like trimer alone is not sufficient to increase the breadth of NAb responses.
Recently, it has been shown that immunization of cows readily generates broad NAbs, likely as a result of their unique germ line antibody repertoires (54). We believe that guinea pigs are a better model for predicting immunogencity in primates and humans, as studies with both foldon and SOSIP Env gp140s have shown comparable antibody profiles in guinea pigs and primates (32,55).
Future immunogen design strategies will likely need to include modifications to limit V3 exposure, as well as to prime neutralizing responses to a greater number of epitopes. A variety of strategies exist to minimize V3 exposure, including using a SOSIP Env immunogen (27,28,56). These immunogens, however, elicit minimal heterologous tier 2 NAbs despite being accurate mimics of the HIV-1 Env trimer. Therefore, rational immunogen designs paired with SOSIP trimers may be beneficial for improving tier 2 NAb responses to pair stabilizing mutations with improved sequence designs. Concepts to improve neutralization breadth, such as utilizing immunogens that target the germ line precursors, assessing strategies to increase somatic hypermutation, and using bioinformatics approaches to create multivalent immunogen cocktails, paired with longitudinal boosting, should continue to be explored.

MATERIALS AND METHODS
Plasmids, cell lines, protein production, and antibodies. The codon-optimized synthetic genes for all HIV-1 Env gp140s were produced by GeneArt (Life Technologies). All constructs contained a consensus leader signal sequence peptide, as well as a C-terminal foldon trimerization tag followed by a His tag (32,57).

Long-Term HIV-1 Env Vaccine in Guinea Pigs
Journal of Virology  (20,21,33). His-tagged proteins were purified by using a HisTrap Ni-nitrilotriacetic acid (NTA) column (GE Healthcare). Ni-NTA columns were washed with 20 mM imidazole (pH 8.0), and protein was eluted with 300 mM imidazole (pH 8.0). Fractions containing protein were pooled and concentrated. Protein constructs were further purified utilizing gel filtration chromatography on Superose 6 (GE Healthcare) in running buffer containing 25 mM Tris (pH 7.5) and 150 mM sodium chloride. Purified proteins were concentrated using CentriPrep YM-50 concentrators (Millipore), flash frozen in liquid nitrogen, and stored at Ϫ80°C.
Guinea pig vaccinations. Outbred female Hartley guinea pigs (Elm Hill) were used for all vaccination studies and were housed at the Animal Research Facility of Beth Israel Deaconess Medical Center under approved Institutional Animal Care and Use Committee (IACUC) protocols.
Guinea pigs (n ϭ 5/group) were immunized with Env protein intramuscularly in the quadriceps bilaterally at 4-week intervals, with two long-term rests, for a total of 12 injections. Vaccination groups included C97ZA012 gp140 only (C97, n ϭ 5); a sequential prime/boost regimen, including clade C C97ZA012, clade A 92UG037, Mosaic 3.1, and clade B PVO.4 gp140s (ABCM, n ϭ 5); and clade C C97ZA012, 459C, 405C, and 939C gp140s (4C, n ϭ 5) (Fig. 1A). For ABCM, one animal died following blood draw and was excluded from the analysis. One guinea pig died at week 134 and one at week 177 in the C97 vaccination group due to age.
Twelve vaccinations were given as three sets of 4 immunizations at weeks 0, 4, 8, and 12, at weeks 62, 66, 70, and 74, and at weeks 104, 108, 112, and 116 (Fig. 1A). Animals were followed for a total of 200 weeks. Vaccinations consisted of a total of 100 g of Env gp140 per injection formulated in 15% Emulsigen (vol/vol) oil-in-water emulsion (MVP Laboratories) and 50 g CpG (Midland Reagent Company) as adjuvants. Serum samples were obtained from the vena cava of anesthetized animals. Endpoint ELISAs. Serum binding antibodies against gp140 and V1/V2 scaffolds were measured by endpoint enzyme-linked immunosorbent assays (ELISAs) (32). Briefly, ELISA plates (Thermo Scientific) were coated with individual gp140s or V1/V2 scaffolds and incubated overnight. For isotype and subclass ELISAs, plates were coated with C97ZA012 gp140. Guinea pig sera were then added in serial dilutions and detected with a horseradish peroxidase (HRP)-conjugated goat anti-guinea pig secondary antibody for total IgG ELISAs (Jackson ImmunoResearch Laboratories). For isotyping ELISAs, HRP-conjugated goat anti-guinea pig IgG1, goat anti-guinea pig IgG2, goat anti-guinea pig IgM, and sheep anti-guinea pig IgA were utilized (MyBiosource). Plates were developed and read using the SpectraMax plus ELISA plate reader (Molecular Devices) and SoftMax Pro 4.7.1 software. Endpoint titers were considered positive at the highest dilution that maintained an absorbance that was Ͼ2-fold above background values.
Avidity ELISAs. Serum binding antibody avidities to HIV-1 Env gp140 were measured by a urea disruption enzyme-linked immunosorbent assay (ELISA) (58). Briefly, ELISA plates (Thermo Scientific) were coated with individual Env gp140s and incubated overnight. Guinea pig sera were prepared in a dilution plate to values between 1.0 and 1.5 at optical densities of 450 nm (OD 450 ) to OD 800 to provide antibody concentrations within a linear range. Sera were plated in duplicate twice; one duplicate was treated with 8 molar urea (Sigma-Aldrich) and the other with distilled water as a control. Plates were then incubated with an HRP-conjugated goat anti-guinea pig secondary antibody (Jackson ImmunoResearch Laboratories). Plates were developed and read using the SpectraMax plus ELISA plate reader (Molecular Devices) and SoftMax Pro 4.7.1 software (OD 450 to OD 800 ). Percent avidity was calculated using the following formula: [(average absorbance of urea-treated sample/average absorbance of water-treated, matched sample) ϫ 100]. The avidity index describes 0 to 30% as low-avidity, 30 to 50% as moderate-avidity, and Ͼ50% as high-avidity binding antibodies.
Competition ELISAs. NAbs PG9, 3BNC117, PGT121, and 447-52D IgG were biotinylated using the EZ-Link micro NHS-PEG 4 (N-hydroxysuccinimide ester-4-unit polyethylene glycol) biotinylation kit (Thermo Scientific) following the manufacturer's instructions. Antibodies were used at readings of approximately 1.0 to 1.5 at OD 450 to OD 550 for each coating protein. For PG9 IgG competition, Mosaic 3.1 Env gp140 was utilized as the coating protein, and for 3BNC117, PGT121, and 447-52D IgG competition, C97ZA012 Env gp140 was utilized as the coating protein. ELISA plates (Thermo Scientific) were coated overnight with gp140s. Guinea pig sera in blocking buffer (1% bovine serum albumin, 1ϫ phosphatebuffered saline, 0.05% Tween) were added at a 1:10 dilution and serially diluted at 1:3 dilutions down the plate. Week 0, 16, 78, 120, 138, and 200 samples were run side-by-side on the same plate. A single biotinylated monoclonal IgG was then added at a single concentration. Streptavidin-HRP (Thermo Scientific) in blocking buffer was then added. Plates were then developed with SureBlue tetramethylbenzidine (TMB) microwell peroxidase substrate and TMB stop solution (Kirkegaard & Perry Laboratories, Inc.). Plates were developed and read at OD 450 to OD 550 using the SpectraMax plus ELISA plate reader (Molecular Devices) and SoftMax Pro 4.7.1 software.
Peptide microarrays. Peptide arrays were generated, assays conducted, and data analyzed using described previously methods (39). RepliTope antigen collection HIV ultra slides (JPT Peptide Technologies GmbH) were utilized. Each slide contains 6,654 15-mer peptides, printed in triplicate (subarrays), overlapping by 11 amino acids, representing 135 different clades or circulating recombinant forms (CRFs) that cover the entire HIV-1 genome. Microarray slides were incubated with guinea pig sera diluted 1/200 in SuperBlock T20 (Tris-buffered saline [TBS]) blocking buffer (Thermo Scientific). Alexa Fluor 647-conjugated AffiniPure goat anti-guinea pig IgG(HϩL) was used as the secondary detection antibody (Jackson ImmunoResearch Laboratories). All batches of slides with the same time point were run in parallel with a control slide incubated with the secondary antibody only.
Slides were scanned with a GenePix 4300A scanner (Molecular Devices), using 635-nm and 532-nm lasers. The fluorescence intensity for each feature (peptide spot) and threshold values for positivity were calculated using GenePix Pro 7 software and GenePix Array List as described previously (39,59). The fluorescence signal from a control slide incubated with just the secondary fluorophore was subtracted from the fluorescence signals from all experimental slides to remove the background signal associated with the fluorophore. For each batch of slides run together, the highest P value from all arrays run that was Ͻ10 Ϫ16 was chosen as the cutoff for all slides for stringency (21,60). All P values that fell below the cutoff P value of Ͻ10 Ϫ16 were set to equal zero, and all samples with P values greater than the cutoff value were maintained as their raw, positive signal. Statistically significant differences between the results for guinea pigs that received the C97 prime/boost regimen and for animals that received either the ABCM or 4C prime/boost regimen at a matched time point (P Ͻ 0.05, Mann-Whitney U test) were determined using Prism 7.0 (GraphPad).
TZM.bl neutralization assay. Functional neutralizing antibody responses against HIV-1 Env pseudoviruses were measured using a luciferase-based virus neutralization assay in TZM.bl cells as described previously (42). Murine leukemia virus (MuLV) was included in all assays as a negative control. For graphing tier 1 pseudovirus data, responses were considered positive if they were greater than the MuLV value and were greater than 0 after MuLV background subtraction. Pseudoviruses were prepared as described previously (42,61). Statistical analyses were conducted on tier 1 pseudovirus neutralization responses utilizing Prism 7.0 (P Ͻ 0.05, Mann-Whitney U test) (GraphPad).
For assessing neutralization against tier 2 pseudoviruses, polyclonal IgGs were purified from sera to reduce the background signal. High-capacity protein A agarose (Thermo Scientific) was used, following the manufacturer's instructions, and buffer exchanged into 1ϫ phosphate-buffered saline (PBS), pH 7.4 (Gibco). Purified polyclonal IgGs were used in the TZM.bl assay against the global tier 2 panel (44) at week 120, which was the terminal peak time point. As animals only showed positivity against X1632 and 25710, purified polyclonal IgGs from the remaining peak time points were run against select tier 2 pseudoviruses. Statistical analyses of tier 2 neutralization responses were conducted utilizing Prism 7.0 (P Ͻ 0.05, Student's t test) (GraphPad).
Antibody-dependent guinea pig neutrophil phagocytosis. C97ZA012 gp140 was biotinylated and coupled to red fluorescent beads (Thermo Scientific). Sera from vaccinated or naive guinea pigs were diluted 1:12,500 in Roswell Park Memorial Institute (RPMI) 1640 medium and 10% fetal bovine serum (FBS) culture medium and incubated with gp140-coupled beads for 2 h at 37°C. Guinea pig white blood cells were isolated from EDTA-treated whole blood from naive guinea pigs. Red blood cells were lysed in ammonium chloride-potassium (ACK) lysis buffer, and white blood cells were collected by centrifugation, washed 3 times with 1ϫ PBS, and resuspended to a volume of 5 ϫ 10 5 cells/ml in RPMI 1640 -10% FBS culture medium. Diluted serum and bead complexes were pelleted at 1,000 rotations per minute for 10 min, the supernatant removed, and 5 ϫ 10 4 cells/well of guinea pig white blood cells were added and incubated for 1 h at 37°C. Following incubation, cells were washed with 1ϫ PBS and stained with blue viability dye for 15 min at 4°C. Cells were stained with anti-CD11R1 antibody (clone MIL4; Bio-Rad) and anti-CD4 antibody (clone CT7; Bio-Rad) for 15 min at room temperature. Cells were washed twice with 1ϫ PBS, fixed with 4% paraformaldehyde, and analyzed for phagocytosis by flow cytometry (BD LSR Fortessa and FACSDiva). Neutrophils were defined as side scatter high (SSC high ), CD11R1 ϩ , and CD4 Ϫ as described previously (63), using FlowJo analysis software, and a phagocytic score was determined using the following formula: (% bead-positive SSC high , CD11R1 ϩ , and CD4 Ϫ cells ϫ MFI of bead-positive cells)/10,000, where MFI is mean fluorescence intensity.


s2fieldsofstudy: Biology Medicine

text:
MicroRNA-365 suppresses cell growth and invasion in esophageal squamous cell carcinoma by modulating phosphoserine aminotransferase 1

Background A number of studies have indicated that expression of miRNA-365 (miR-365) is suppressed in various cancers, suggesting its cancer-suppressive role. In the present investigation, we evaluated the regulation and character of miR-365 in human esophageal squamous cell carcinoma (ESCC). Patients and methods The tumor tissues and adjacent nontumor tissue samples were collected from 30 patients having ESCC, and the expression levels of miR-365 were studied by quantitative real-time polymerase chain reaction (PCR). MTT and cell invasion by Matrigel assay were done to study the effect of miR-365 on proliferation and metastasis of ESCC cells. An in vivo tumor model was generated by inoculating ESCC cells subcutaneously into BALB nude mice. A study of various biomarkers such as quantitative polymerase chain reaction (qPCR), luciferase activity assay, and Western blot was done to confirm the targets of miR-365. Results In tumor tissues, a significant downregulation of miR-365 was observed versus the nontumor adjacent tissues and ESCC cells versus the selected esophageal endothelial cells. It was observed that higher expression levels of miR-365 inhibited the cell invasion, colony formation, growth in esophageal cancer cell lines in vitro, and tumor development in vivo. The study of biomarkers suggests involvement of phosphoserine aminotransferase 1 (PSAT1) as a favorable target for miR-365, and its abnormal expression inverted the miR-365-arbitrated suppression of invasion, viability, and epithelial–mesenchymal transition in esophageal cancer cells. A negative correlation existed with expression of miR-365 and PSAT1 in human esophageal cancer tissue samples. Conclusion The study established that miR-365 exhibits tumor-suppressive action via regulating the levels of PSAT1 and leads to invasion and progressiveness of esophageal cancer.

Introduction
Esophageal squamous cell carcinoma (ESCC), which also includes esophagus adenocarcinoma, ranks sixth among all types of cancers causing mortality and eighth in the list of common cancers worldwide. 1,2 ESCC is reported to be the most happening cancer (70%) among all the reported cases of esophageal cancer worldwide, and in China, the figures are even more alarming as the occurrence of ESCC is at least 30 times more compared with that in USA. 3 ESCC originates from the esophagus, mostly from the epithelial cell linings. The etiology mainly associated with the occurrence of ESCC includes smoking, poor diet plan, alcoholism, and hot drinks. 1 The high mortality rate in ESCC is mainly due its late diagnosis and its aggressive nature.

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Sun et al miRNAs are noncoding RNA molecules having single strand (23 nucleotides), and these molecules can bring about regulation of gene expression via binding to the 3′-untranslated regions (UTRs) of target genes at the posttranscriptional level. 4,5 Recently, the human genome has been reported to show presence of a large number of miRNAs (>1000) that regulate ~30% of all the discovered genes. 6 A number of studies have indicated that deviant levels of miRNAs are linked with disorders such as cancer; based on the type of mRNAs they can act as tumor suppressors. [7][8][9][10][11] The miRNA-365 (miR-365) is confirmed for its tumor-suppressive effect in a number of cancers such as cutaneous squamous cell carcinoma, 12 lung, 13 breast, 14 and hepatic carcinoma. 15 ESCC has been reported for its aberrant expression of miRNAs identified using quantitative reverse transcription polymerase chain reaction (qRT-PCR); 16,17 however, studies suggesting the correlation between expression of miR-365 and ESCC along with specific mechanisms of miR-365 in development of esophageal cancer have not been reported yet.
In cancer, the cells tend to grow rapidly and hence alter metabolic pathways, and to meet these cell requirements, metabolic pathways affecting the production of glycine and serine play an important role. 18 Recently, a number of tumorigenesis enzymes have been identified, which are involved in biologic reactions facilitating to maintain the redox status and the biosynthesis of cellular components. 19,20 Among them, phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH) are the two enzymes responsible for the catalysis of serine. These enzymes are found to be upregulated in a number of cancers and are reported to be responsible by diverting the glucose flux for metabolism of serine. 20 However, the regulatory mechanism behind the expression of these enzymes and their role in cell proliferation are still not clearly understood.
In the present research, we first established that expression of miR-365 was significantly downregulated in ESCC cell lines and tissues. Our study further confirmed the tumorsuppressive activity of miR-365 in ESCC via the PSAT1 pathway. The outcomes of our study confirmed a new therapeutic target for ESCC.

Patients and methods
Human esophageal tissue specimens A total of 30 patients diagnosed with esophageal carcinoma in 2016 and who underwent surgery for esophageal resection before submitting to chemotherapy at Subei People's Hospital of Jiangsu Province were selected for this study. From these patients, tissue specimens of ESCC and the adjacent area (normal tissue) were collected after surgery under the guidelines from an experienced pathologist. Before storing in liquid nitrogen, the tissues were dissected to remove any connective tissue. The tissue samples were stored in the tissue bank of Subei People's Hospital of Jiangsu Province, and on receiving, the samples were coded in accordance with the guidelines from the ethical committee. All the protocols were in accordance with the local human ethical committee of Subei People's Hospital of Jiangsu Province followed by the approval from hospital board (sanctioned reference number HEC/SPHJ/ESO/14B-16/17). Written informed consent was obtained from all patients.

Cell lines and culture conditions for in vitro studies
The human esophageal cancer cell lines were procured from Cell Collection Center, Shanghai (Chinese Academy of Science-China). Three ESCC cell lines (EC109, EC1, and EC9706), HEEC, and HEK293T cells for transfection were used in the present study and were obtained from Chinese Academy of Medical Science (Shanghai, China); the cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 units/ mL penicillin G at 37°C and 5% CO 2 . The cells were grown in EpiCM-2 along with EpiCGS-2 (Innoprot, Derio, Spain).

Extraction of RNA and qRT-PCR studies
Total miRNA was isolated from the cells using an miRNeasy kit (Qiagen GmbH, Hilden, Germany) in accordance with the manufacturer's protocol; next, an miScriptRT kit was used to convert it into cDNA. RNase-free water was used to elute RNA, followed by recording the absorbance at a wavelength of 280 nm, and then used for reverse transcription. TaqMan assay was done by using TaqMan Human miRNA Array Panel (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions, and the expression was normalized using endogenous U6. The miR-365 mimics and negative control RNA were from GenePharma (Shanghai, China). For transcription of PSAT1, iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used as per the manufacturer's instructions for synthesizing cDNA. Real-time qPCR was done using an ABI 7,500 Real-Time PCR System (Thermo Fisher Scentific). The stem-loop RT primer, qRT-PCR primers, and the internal control U6 primers were used (Table 1). U6 was opted as internal control for relative expression of miR-365. Each trial was performed three times.

Construction of plasmid and transduction of lentiviral and gene transfection
To construct the miR-365, lentiviral gene transfer vector carrying hsa-miR-365, amplification of fragment-encoding sequence for pre-miR-365 was done by PCR followed by cloning into the sites of pGCsil-green fluorescence protein (GFP) vector (Shanghai Genechem Co., Ltd, Shanghai, China) to get the lentivirus-mediated miR-365 vector (LV-miR-365). To generate the PSAT1 overexpression plasmid, a pcDNA3 vector was used. The amplification of PSAT1 cDNA was done by PCR using cDNA extracted from HL60 cell lines. After amplification, the PSAT1 cDNA gene was cloned into the vector.
The 3′-UTR fragment of PSAT1 having the recognition sequence of miR-365 was subjected to amplification using PCR using the required primers. After amplification, subcloning was done into the vector (psiCHECK-2; Promega Corporation, Fitchburg, WI, USA) to construct PSAT1 3′-UTR clone using Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific). The pCDH cDNA was transfected in the PSAT1 packaging system for generating virus particles using Lipofectamine 2000 reagent (Thermo Fisher Scientific) into HEK293T cells as per the manufacturer's instruction. The cells, after growing to the log phase, were transfected with pGCsil-GFP vector for 24 h. Mass screening of stable cells was done after looking into the levels of GFP mediated by lentivirus vector post 72 h of transfection using a flow cytometer (BD Biosciences, San Jose, CA, USA).
For carrying out gene transfection, the ESCC cells (5×10 5 ) were transfected in 2 mL culture medium with 100 pmol control vector in a serum-free medium with Lipofectamine reagent. The culture medium was changed after 6 h, and the cells were maintained in an incubator at 37°C in 5% CO 2 for 48 h.

3′-UTR dual-luciferase reporter assay and the human embryonic kidney cells 293
To build the luciferase reporter vector, the binding sites for miR-365 in the PSAT1 and the PSAT1 3-UTR fragment were amplified by PCR using the reverse primers. The amplified protein was then inserted into the luciferase gene psiCHECK2 vector, named as PSAT1 3′-UTR-wild. Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific) was used for site-directed mutagenesis of miR-365 of the target site in PSAT1 3′-UTR, named as PSAT1 3′-UTR-mut. The transfection was done with miR-365 mimics and with either PSAT1 3′-UTR-mut or wild. The luciferase assay was done 48 h post-transfection using Luciferase Reporter Gene Detection Kit (Sigma-Aldrich Co., St Louis, MO, USA).

Cell invasion assay
For the assay, a Matrigel-coated (1:5 serum-free RPMI medium-1640) membrane filter (8 μm) was used. The membrane filter was subjected to sterilization and air-drying, and then 20 μL of medium was added into the bottom chamber of corning Transwell ® plates. The transfected cells (1.5×10 6 cells/mL) were resuspended in serum-free RPMI medium and were subjected to seeding in the upper space of Transwell ® plates followed by incubation for 24 h at 37°C. The cells on the bottom of the filter were fixed in methanol at −20°C for 15 min followed by washing with PBS and later staining of crystal violet, and the cells were counted under a microscope.

Cell viability studies (MTT assay)
The in vitro cell viability of EC109 and EC9706 cells was evaluated by MTT assay using Vybrant ® MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific) following the manufacturer's protocol. The procedure involved replacement of medium with fresh medium containing MTT (0.5 mg/ mL) at defined time intervals indicated by the manufacturer. The cells were then subjected to incubation for 1 h at 37°C resolved by the provided reagent followed by measurement of absorbance at 570 nm.

Colony formation assay
The EC109 and EC9706 cells were trypsinized and suspended and cultured in culture plates (60 mm diameter) along with 10% FBS for 2 weeks. The staining and fixing of developed colonies were done using 0.5% crystal violet solution for 20 min followed by washing (three times). Clusters with cell count >50 were counted as colonies. Microscopic examination and counting of colonies were done from 10 randomly selected viewed fields, and the mean representing the 95% confidence region was attained.

Expression of proteins by Western blotting
After receiving the treatment, the cells were subjected to lysis using RIPA buffer (Cell Signaling Tech, Inc, Danvers, MA, USA), and the extracts were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes and blotting. The membranes were blocked using a mixture of 5% skim milk and 0.1% Tween 20 in Tris-buffered saline. The membranes were incubated with I ry antibodies at 4°C for 12 h. PSAT1, p-GSK-3 β, Snail, E-cadherin, GSK-3β, Vimentin (Thermo Fisher Scientific), and the anti-GAPDH antibody were used as internal controls (Thermo Fisher Scientific). The blots were visualized using enhanced chemiluminescence after incubating with secondary antibodies for 2 h.

Statistical data analysis and significance
All the data reported were average±SD (n=3). One-way analysis of variance or t-test (paired) was used for establishing differences. The values of p <0.05 were regarded as statistically significant. The expression of relative proteins by Western blotting was done, using GAPDH as loading control.

Results
Levels of miR-365 are decreased in EC1, EC109, and EC9706 cell lines and tissues The esophageal cancerous tissues and the proximate noncancerous tissues in the same subjects were obtained from esophageal cancer patients (30 numbers), and the tissues were subjected to qRT-PCR for comparing the expressions of miR-365. The outcome of the experiment showed that the expression of miR-365 was significantly (P<0.01) toward lower side in tumor tissues recovered from patients compared to nontumor adjacent tissues ( Figure 1A). The miR-365 levels in ESCC cells, that is, EC109, EC1, and EC9706, were similar to those observed in tissue samples, and the levels of miR-365 were on the higher side when compared with the normal HEEC cell lines. The expression levels were toward the higher side in EC109 and the lowest in EC9706 ESCC cell lines ( Figure 1B); hence, EC109 and EC9706 were selected for experiments in the study.

MiR-365 suppresses the growth of ESCC cells (in vivo and in vitro)
To study the effect of miR-365 on esophageal cancer in vivo and in vitro, the EC9706 and EC109 cells were transfected with overexpression vector of miR-365, and the cell viability and tumor growth were evaluated. qRT-PCR was utilized for confirming the overexpression levels of miR-365 in EC109 and EC9706 cell lines (Figure 2A) 2B and 3A). Tumor growth was evaluated in the xenografts mouse model, and the tumor weight reduced significantly (P<0.01) ( Figure 3B) in mice receiving injection of EC9706 cells overexpressed for miR-365 compared with those receiving injection of vector control cells.

Inhibitory action of miR-365 on cell invasion in ESCC cell lines
Cell invasion or Matrigel invasion assay was done to analyze the role of miR-365 vector transfection in esophageal cell lines. The results demonstrated that miR-365 overexpression vector suppressed the invasion of selected EC9706 and EC109 cell lines significantly (P<0.01) by ~64% compared to control ( Figure 4A and B).

Identification of PSAT1 as a favorable target site of miR-365
To identify the molecular mechanism behind miR-365, the experiments using prediction tools were designed to examine the potential targets. A prediction algorithm of Targetscan To establish this prevision, luciferase activity was evaluated to validate PSAT1 as a target for miR-365. The constructed luciferase reporter plasmids built by cloning the mutant 3′-UTR of PSAT1-expressing mutations in the miR-365 overexpression vector and the miR-365 seed region were used to co-transfect the HEK293 cells. On assessing the luciferase activity, it was found that the overexpression of miR-365 did not suppress the reporter gene activity for mutations of PSAT 3′-UTR on two binding sites but significantly suppressed the activity of the wild-type PSAT1-UTR-MUT1 or MUT2 (P<0.01) ( Figure 5A). The abnormal expression of miR-365 decreased the mRNA expression of PSAT1 significantly compared to the loading control, that is, GAPDH in both the ESCC cell lines ( Figure 5B), confirming PSAT1 as a favorable target for miR-365. The outcomes of miR-365 on tumor invasion were analyzed by expression of epithelial markers such as E-cadherin, Snail, Vimentin, the Ser/Thr kinase GSK-3β, and EMT, that is, epithelial-mesenchymal transition markers. Reports suggest a correlation between the transcription factors Snail and EMT; Snail modulates the levels of E-cadherin, whereas downregulation of E-cadherin stimulates the activity of EMT. Reports have established that Ser/Thr kinase family protein GSK-3β is deactivated as a result of phosphorylation at Ser9. GSK-3β is found to regulate Snail activity; also, the epithelial phenol-typing is maintained by suppressing Snail, and hence, deactivation of GSK-3β elevates EMT. 21 The results of Western blot as depicted in Figure 5C suggested that overexpression of miR-365 increased the expression levels of E-cadherin, suppressed the levels of Snail as well as Vimentin, and decreased the levels of total and associated phosphorylated GSK-3β followed by decrease in the levels of PSAT1. The results of overall protein expression suggested that miR-

Involvement of PSAT1 in miR-365mediated suppression of cell proliferation and invasion in ESCC cell lines
To further confirm the role of miR-365-mediated suppression of PSAT1, in correlation to proliferation and invasion of ESCC cell lines. The study showed that the expression of PSAT1 was ectopic, which in turn partly reclaimed the suppression of cell invasion and proliferation caused by overexpression of miR-365 in ESCC cell lines ( Figure 6A and B). The study also showed that the ectopic expression of PSAT1 resulted in partial restoration of the E-cadherin and Snail levels, which were partially suppressed by miR-365, confirming the role of PSAT1 in reversing the consequences of miR-365 on EMT in selected esophageal cell lines ( Figure 6C). A negative correlation was established between levels of mRNA of PSAT1 and expression of miR-365 by qRT-PCR studies involving miR-365 and levels of PSAT1 in cancerous tissue samples from patients reported with esophageal carcinoma (P<0.01, R 2 =0.369). The study established that miR-365 is involved in the regulation and expression of PSAT1 in ESCC (Figure 7).

Discussion
Here, we studied the role and extent of expression of miR-365 in ESCC and also the mechanism involved. We demonstrated that ESCC (cell lines and ESCC-affected human tissue) is associated with the downregulation of miR-365, and its overexpression halts its progression. Our findings suggested Esophageal cancer is colligated with both inadequate prognosis and survival. 22,23 Advanced-stage diagnosis has attributed to the poor survival of ESCC patients, thus creating the necessity to discover specific biologic markers for its early-stage detection. A number of reports have worked on discovering miRNAs for distinguishing ESCC from normal tissues using microarray analysis.
In a study involved in the microarray analysis of human miRNAs in cancerous ESCC tissues, out of 210 miRNAs, about four, that is, miR-373, let-7d, miR-340, and miR-330, were upregulated. 16 25 Similarly, a study involved in identifying miRNAs reported 31 miRNAs in correlation with ESCC, 26 among which seven were able to distinguish ESCC from the normal tissues. The study confirmed that the expression levels of miR-140, -29c, -99a, and -100 were downregulated, while those of miR-151, miR-424, and miR-25 were upregulated. 26 Yan et al 27 reported decreased expression of miR-340 in ESCC and found that miR-340 caused inhibition by targeting PSAT1. 28 Although a number of studies have dug into the role of miRNAs in the progression of ESCC, the identified potential targets need further confirmation. Kano et al found that the three miRNAs (miR-133a, mir-133b, and miR-145) upon transfection into ESCC cells caused inhibition of cell proliferation and invasion. 25 In a study related to ESCC, it was found that miR-25 inhibited cadherin 1 activity. 29 Similarly, many potential targets were identified in other studies involving ESCC, which include the ERK5 pathway for miR-143, ZEB1 for miR-150, Rap1b for miR-518b, and FBXW7 for miR-223. 30 In the present investigation, we established that miR-365 is downregulated in esophageal tumor tissues and ESCC cell lines, and we also evidenced that the expression of miR-365 is linked to proliferation, and colony-forming and invasion capability of cancerous esophageal cells in addition to the growth of tumor in mice. The study established that miR-365 exhibited tumor-suppressing activity in esophageal cancer by aiming the PSAT1 pathway. The outcome of experiments demonstrated that the overexpression of miR-365 resulted in the downregulation of PSAT1, and the abnormal expression of PSAT1 reversed the inhibitory activity of miR-365 on cell proliferation, invasion, and expression of EMT.
PHGDH and PSAT1 are the enzymes of serine pathway; literature suggest modifications in the levels of these two associated with progression of many cancers such as breast cancer. 31 The expression of PHGDH is reported to be increased in some cases of breast cancer, and the downregulation of PSAT1 exhibited antiproliferative effect in such PHGDHoverexpressing breast cancer cells. 32,33 The overexpression of PSAT1 in colon cancer is associated with chemoresistance. 31 A weakened response for endocrine treatment approach in breast cancer is linked to overexpression of PSAT1. 33 All the reports confirmed participation of PSAT1 in progression of cancer, which adheres to our present findings suggesting that suppression of miR-365 may result in tumorigenesis in esophageal cancer by upregulation of PSAT1. In future, experiments focusing on the correlation between PSAT1 and miR-365 may further strengthen their roles in treating ESCC.

Conclusion
The outcomes of the present study confirmed tumor suppressor effect of miR-365 in ESCC by modulating the expression levels of PSAT1. The study confirmed that the effect of miR-365 on cell invasion and proliferation gets reversed after abnormal expression of PSAT1. Hence, this study confirmed that miR-365 exerts antitumor effect via PSAT1 pathway and hence suggests a novel biomarker in the development and progression of ESCC.

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