extfieldsofstudy: Biology Medicine

text:
DGKZ Acts as a Potential Oncogene in Osteosarcoma Proliferation Through Its Possible Interaction With ERK1/2 and MYC Pathway

Osteosarcoma (OS) is one of the most common primary bone tumors in children and young adults. The majority of osteosarcoma patients have limited alternative therapeutic options and metastatic patients generally have a poor prognosis. Thus, it is important to explore novel effective therapeutic targets in the treatment of osteosarcoma. Diacylglycerol kinase zeta (DGKZ) is a recently identified gene potentially associated with certain human carcinogenesis. However, the role of DGKZ in proliferation of osteosarcoma is still unclear. In this study, DGKZ's expression was firstly investigated in OS tumor samples and correlated with poor outcome in OS patients. Silence of DGKZ by shRNA hampered osteosarcoma cell growth and promoted cell apoptosis in vitro. In vivo, DGKZ's knockout also suppressed xenograft tumor proliferation as determined by bioluminescence imaging and weight/volume measurements. Meanwhile, Affymetrix GeneChip and Ingenuity Pathway Analysis (IPA) revealed that DGKZ knockdown resulted in a decreased activity of MYC pathway, and several target genes expression in MYC pathway were altered, including CCND1, CDKN2B, CDK6, PCNA, and EGR1. Furthermore, immunoprecipitation coupled with mass spectrometry (IP-MS) analysis was used to identify proteins that interacted with DGKZ in OS cells and revealed ERK1/2, a key MYC-interactor, to associate with DGKZ. Together, our study demonstrated that DGKZ might act as an oncogene in osteosarcoma via its possible interaction with ERK1/2 and MYC pathway.

INTRODUCTION
Osteosarcoma is the most common aggressive form of bone tumor occurs in children and young adults (1,2). Traditional therapeutic approaches include local control of the primary lesion by surgery and/or chemotherapy, and treatment of disseminated disease with multiagent cytotoxic.
Chemotherapy (3), Somehow, patients those with metastatic or recurrent disease have extremely poor survival rates even after normative chemotherapy and curative resection of the primary lesion (4)(5)(6). Hence, there is urgent need for development of new therapeutic approaches and a further investigation in proliferation of osteosarcoma. However, progression of osteosarcoma is associated with numerous genes with diverse genomic alterations and the molecular carcinogenesis of osteosarcoma has not been fully elucidated (7). Diacylglycerol (DG) is an important second messenger involved in a variety of cellular responses including proliferation, differentiation, motility, and secretion (8,9) which could activate various effector proteins. Intracellular DAG levels have been shown to play a vital role in cellular growth responses (10). Thus, levels of DG should be maintained strictly to keep cellular environment within a physiological range. Diacylglycerol kinases (DGK) are a class of enzymes that convert DAG into PA and act as key modulators of DG levels (11). Recent studies have found 10 isozymes of DGK, each showing unique tissue expression and distinct subcellular localization (12)(13)(14)(15).Among the 10 isoforms, diacylglycerol kinase zeta (DGKZ) is the only one that contains a nuclear localization signal (16,17) and has proven to be associated with various physiological or pathophysiological signaling pathways (16,(18)(19)(20)(21)(22)(23)(24)(25). Recent studies have proven that in colon cancer and gliomas, DGKZ plays an key role in oncogenesis. (26)(27)(28). Additionally, DGKZ also showed its function in regulating bone homeostasis through osteoclasts via modulation of c-Fos (20), which is a key regulator in proliferation of osteosarcoma (29)(30)(31)(32). However, the potential role of DGKZ in oncogenesis of osteosarcoma is still unclear.
In our study, DGKZ's expression was upregulated in osteosarcoma tissues and correlated with patients' poor prognosis. In vitro, DGKZ knockdown impaired proliferation and promoted apoptosis in osteosarcoma cells. In vivo, DGKZsilencing xenografts in nude mice formed smaller tumors while compared with control group. Additionally, microarray and IPA analyses demonstrated a alternation in expression of signaling-related genes in osteosarcoma cells and verified MYC pathway as a direct downstream target of DGKZ. In addition, association between DGKZ and ERK1/2, a key MYC-interactor, was verified through immunoprecipitation-mass spectrometry (IP-MS) approach. These results suggest a potential interaction between DGKZ and ERK1/2-MYC pathway might play an important role in promotion of osteosarcoma, indicating that interfering with function or expression of this interaction may be a potential route to block the invasiveness of osteosarcoma.

Ethics Statement
The Ethics Committees of Affiliated Sixth People's Hospital, Shanghai Jiaotong University has approved all studies about human participants. Informed and written consents were obtained from all patients or their advisers according to ethics committee guidelines.

Patient Samples and Follow-Up
Tumor specimens from 80 patients suffered from IIB limb osteosarcoma between 2012 and 2015 and 24 cases of normal bone tissues as control group were collected from Affiliated Sixth People's Hospital, Shanghai Jiaotong University for this study. The normal bone tissues were resected within at least 5 cm to the margin of tumor while definitive surgery was administrated. After surgical resection, all the osteosarcoma and normal bone tissues were frozen within 30 min. All patients were treated with combination chemotherapy which consist high doses of methotrexate, cisplatin, ifosfamide, and doxorubicin, then underwent local resection of the primary tumor, followed by adequate cycles of adjuvant chemotherapy. After completion of chemotherapy, patients were followed every 3 months for 4 years and then every 6 months for 10 years.

Immunohistochemical Staining of DGKZ in OS Samples
Paraffin sections were treated with hydrogen peroxide to inactivate endogenous peroxidases. Antigen retrieval was performed in a microwave in 10 mmol/L citrate buffer at pH 6.0. Sections were then incubated in anti-DGKZ antibody overnight at 4 • C, and a secondary antibody was used to detect protein expression (27). For each run of immunohistochemistry, appropriate positive and negative controls were performed. Immunohistochemistry staining was assessed by two blinded independent pathological observers who were blinded to patient identity. Percentage of osteosarcoma cells stained positive for DGKZ was recorded, with 0 denoting none of osteosarcoma cells stained, 1 denoting 1-25% of osteosarcoma cells stained, 2 denoting 26-50% of osteosarcoma cells stained, 3 denoting 51-75% of osteosarcoma cells stained, and 4 denoting 76-100% of osteosarcoma cells stained. Intensity of nuclear/cytoplasm/cytomembrane staining on a scale was also recorded, with 0 denoting no staining, 1 denoting slight/weak staining, 2 denoting strong staining; and 3 denoting intense staining. Score was calculated as percentage of cells stained positive multiplied by intensity of staining, and was defined as positive (score above 6) or negative(score ≤6).

Cells Lines and Cell Culture Conditions
Osteosarcoma cell lines that included SaoS-2, U-2OS, HOS, MG-63, human normal osteoblasts hFOB1.19 cell line and human renal epithelial 293T cell line were obtained from China Center for Type Culture Collection (CCTCC, Shanghai, China). Cells were grown in Dulbecco's modified Eagle's medium, Eagle's minimal essential medium, DMEM-F12 growth medium, and McCoy's 5A medium mixed with 10.0% fetal bovine serum (FBS) that were purchased from Gibco (Gibco, USA). The cells were incubated overnight at 37 • C in a 5% CO2 humidified environment. The cells were trypsinized when they reached 75% confluence and were then used for in vitro and in vivo studies.

Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction
Quantitative real-time RT-PCR was performed in triplicate with an Applied Biosystems Prism 7,500 Fast Sequence Detection System using TaqMan universal PCR master mix according to the manufacture's protocol (Applied Biosystems Inc., Foster City, California, USA). TaqMan probes and primers were purchased from Applied Biosystems Inc. Levels of RNA expression were determined using the 7,500 Fast System SDS software package (version 1.3.1; Applied Biosystems Inc.) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a control for normalization. Primers used here were as follows: GAPDH for: 5 ′ -TGACTTCAACAGCGACAC--CCA-3 ′ ,GAPDH, reverse:5 ′ -CACCCTGTTGCTGTAGCCAAA−3 ′ , DGKZ for: 5 ′ -AGCAAG-CAAGAAGAAGAAGAGG-3 ′ , and DGKZ reverse:5 ′ -GGATTGAGATACCAGAGGAAAGAC-3 ′ . The relative DGKZ expression was normalized to GAPDH, and data analysis was conducted using the comparative CT method.

Cell Proliferation Analysis
Cells in shCtrl and shDGKZ groups were plated in 96-well plates at a density of 2 × 10 3 cells/well and incubated for 1-5 days. Each group contained three wells. At the end of incubation, 10 µl of 5 mg/mL MTT (Genview, USA) was added and incubated for 4 h at 37 • C. The medium was removed and 150 µL DMSO was added. Absorbance was determined with an enzyme-linked immunosorbent assay reader at 595 nm. The cell proliferation curves were drawn according to the absorbance.
Cell proliferation was also recorded and accessed through counting viable cell number with Cellomics Array-ScanTM VTI HCS Reader (Thermo Scientific, Waltham, MA, USA)by manufacturer's instructions. Briefly speaking, cells were cultured at a density of 2 * 10 3 /well in 96-well plates under 37 • C with 5% CO 2 . From the next day, cells with GFP were taken photos and counted each day. Cell proliferation was recorded consecutively for 5 days. Cell growth curves were drawn according to cell numbers.

Cell Apoptosis Analysis
Cell apoptosis was assayed by staining with Annexin V-APC and detected by flowcytometry. Briefly, cells were washed twice with cold PBS and resuspended in 1 × binding buffer. Then 100 µl of solution (about 1 × 10 6 -1 × 10 7 cells) was transferred to a 5 µl Annexin V-APC and 5 µl PI, and incubated for 15 min at room temperature in the dark. Cells were analyzed using flow cytometry. All experiments were performed in triplicate.
The activity of Caspases3/7 in OS cells was detected with Caspase-3/7 Assay Kit (Promega), following the manufacturer's instructions and cell fluorescence intensity at 499 nm was measured by ELISA Tablet counter for quantitative assessment.

Assessment of Tumor Growth Inhibitory Effects of DGKZ -Silencing in a Xenograft Model
The mouse experiments and animal care procedures were approved by the Ethics Committee of Affiliated Sixth People's Hospital, Shanghai Jiaotong University. Four-week-old male BALB/c nude mice were obtained from Shanghai Laboratory Animal Center (Shanghai, China). HOS cells (4 × 10 6 ) stably expressing DGKZ or shDGKZ were suspended in 150 µL of phosphate-buffered saline and inoculated subcutaneously in the right armpit region (n = 10 per group). After 4 weeks, the mice were sacrificed and the tumors were removed for analysis. Tumor volumes were calculated using the following formula six times during observation by Vernier calipers: 3.14/6 × y (length) × x 2 (width). After the mice were killed, the tumors were resected and weighted. In addition, before sacrificed, tumor growth in the living animals was monitored and quantified by luminescence levels. Bioluminescence was measured with the IVIS imaging system (Xenogen Corp., Alameda, CA, USA). All of the images were taken 10 min after intraperitoneal injection of luciferin (Sigma-Aldrich, St. Louis, MO, USA) of 150 mg/kg body weight, as a 60-s acquisition and 10 of binning. During image acquisition, mice were sedated continuously via inhalation of 3% isoflurane. Image analysis and bioluminescent quantification was performed using Living Image software (Xenogen Corp).

Microarray Gene Expression Analysis
After infection with shDGKZ total RNA was extracted from HOS osteosarcoma cells, and 50-500 ng of RNA was used to generate biotin-modified amplified RNA (aRNA) using a GeneChip primeview human Kit (Affymetrix, USA). Data of gene expression was analyzed by GeneChip R PrimeView TM Human Gene Expression Array (Affymetrix, USA; catalog # 901838) which contains probes for 36,000 genes and subsequent analysis was performed using the Affymetrix Expression Console (EC, version 1.1).For the microarray analysis, following filtering by flag signal, the raw data were processed and normalized. Differentially expressed genes were identified with P < 0.05 and fold changes >2. In addition, IPA (Ingenuity Pathway Analysis) was performed which provided more detailed information regarding the pathways in which the target genes were involved.

Affinity Purification of DGKZ Interacting Complexes and LC-MS/MS Analysis
The DGKZ gene fused with a 3 × FLAG tag sequence (Sigma) at its 5 ′ -end was amplified by PCR and inserted into GV367 lentiviral vector (Genechem, Shanghai, China), and the construct was named as p3 × FLAG-DGKZ. HOS cells were plated at the concentration of 4 × 10 5 cells/well in 6-well plate, 2 ml of DMEM medium containing10 µl of 3×FLAG-DGKZ or negative control lentivirus and 2 µl of polybrene(10 µg/µl) was added into the wells. The stable cells expressing 3 × FLAG-DGKZ was validated by immunoblotting using the specific antibodies. The 3 × FLAG-DGKZ interacting complex was purified using the anti-FLAG magnetic beads (Sigma) according to the manufacture's protocol. The procedure of in-gel trypsin digestion and peptide extraction was performed following the standard protocol. LC-MS analysis was performed on a Nano Acquity UPLC system (Waters Corporation, MA, U.S.) connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive) (Thermo Fisher Scientific, Bremen, Germany) equipped with an online nano-electrospray ion source. The Q-Exactive mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Data from both scans were acquired in profile mode. The spectra were recorded with Xcalibursoftware. The raw mass files generated by the Q-Exactive instrument were processed using Proteome Discoverer software (Thermo Scientific, version 1.4) integrated with the MASCOT (Matrix Science, London, UK; version 2.3.2) search engine for protein identification. Data were searched against the Human UniProtKB/Swiss-Prot database (Release 2017_02_19, 20185 entries).To reduce false positive identification results, a decoy database containing the reverse sequences was appended to the database.

Co-immunoprecipitation and Immunoblotting
The procedure of protein isolation and immunoprecipitation was essentially the same as described in the purification of 3 × FLAG-DGKZ complexes for MS identification. The immunoprecipitated protein complexes were eluted by 3 × FLAG peptide. Samples were separated by SDS-PAGE and Frontiers in Oncology | www.frontiersin.org transferred on PVDF membrane (Millipore, 0.2 µm). The membranes were blocked with 5% non-fat milk and probed with specified primary antibody followed by incubation with the secondary antibody conjugated with horseradishperoxidase, and then the ECL substrate was added on membrane and exposed by ECL system.

Statistical Analysis
Patient survival was analyzed by the log-rank test using SPSS 15.0 software. All experiments were performed in triplicates, and the data were expressed as the mean ± SD. Statistical analyses were conducted with Student's t-test. P < 0.05 was considered statistically significant. FIGURE 2 | Lentivirus-mediated DGKZ knockdown specifically inhibited DGKZ expression in osteosarcoma cells: Saos-2, HOS, and U2OS infected with DGKZ -shRNA or shCtrl lentivirus and non-transfected cells examined by fluorescent microscopy and light microscopy at the 3rd day after infection. More than 80% of cells expressed GFP. There was no significant difference between the negative control group and the non-transfected group, indicating the transfection process has no effect on cells growth.

High Expression of DGKZ in Osteosarcoma Samples/Cells and Its Correlation With Poor Prognosis
To initially detect expression of DGKZ in osteosarcoma, we collected 80 osteosarcoma samples and 24 normal bone samples. Among 80 tumor samples, 37 osteosarcoma samples(46.2%) had high expression of DGKZ with a score defined as positive (above 6). Compared with osteosarcoma samples, DGKZ expression was absent in normal bone samples while all bone samples demonstrated a score defined as negative (≤6, Figures 1A-E). Additionally, DGKZ was detected to distribute in cytoplasm. Furthermore, The level of DGKZ expressed in four OS cell lines (SaoS-2, U-2OS, HOS, MG-63) was significantly higher than normal osteoblasts hFOB1.19 ( Figure 1H).
No correlation was observed between age/gender/tumor location/pathological subtype and DGKZ expression ( Table 1). DGKZ expression was associated with a poor prognosis in IIB limb osteosarcoma patients. According to data from follow-up, while evaluating progression-free survival, patients with DGKZ positive expression had a mean survival time of 36.7 months, whereas patients whose samples with DGKZ negative expression had a mean survival time of 42.9 months (P = 0.018, Figure 1F).
In summary, these results demonstrated that OS patients with high DGKZ expression survived for shorter periods than those with low DGKZ expression and DGKZ might played an essential role in the development and progression of osteosarcoma.

DGKZ Silencing Inhibited Growth and Promoted Apoptosis in vitro
According to the above result, we further explored DGKZ biological function through RNA interference. We did Lentivirus-mediated knockdown of DGKZ in Saos-2, U2OS and HOS cells. Fluorescent microscopy and light microscopy demonstrated that more than 80% of cells expressed GFP at the 3rd day after infection (Figures 2A-O). Compared with mock transfected cells, DGKZ's expression was significantly inhibited in cells with shRNA at mRNA level by qPCR analysis, which demonstrated that the lentiviral-based shRNA construct exerted a specific knockdown effect on endogenous DGKZ expression in Saos-2, U2OS, and HOS cells (Figures 2P-R).
Sustaining proliferation signaling is one of the core hallmarks of cancer (33). Thus, we investigated cell proliferation in Saos-2, U2OS, and HOS cells with or without shDGKZ by HCS and  Figures 3, 4, significant decrease in proliferation was observed in shDGKZ group by MTT and HCS assay in all three cell lines.

MTT assays. As shown in
Resisting cell death, another hallmark of cancers, is essential for tumors (34). Thus, we evaluated Caspase3/7 activity in Saos-2, U2OS, and HOS cells with shDGKZ. As demonstrated in Figures 5A-C, Caspase3/7 activity, measured by using a luminescent caspase activity assay kit, increased remarkably in DGKZ-silencing Saos-2/U2OS/HOS cells compared with control group. Annexin V-APC staining by FACS in Saos-2,U2OS and HOS cells following DGKZ -knockdown was also administrated to check impact of GINS1 on apoptosis. Figures 5D-F showed the apoptotic rate in DGKZ-silencing cells was much higher than that in control group in all three cell lines, suggesting that DGKZ knockdown facilitated apoptosis of osteosarcoma cells.

Silencing of DGKZ Inhibits Tumorigenicity in vivo
We next examined whether DGKZ silencing in osteosarcoma affects OS cell growth in vivo. Right armpit region of BALB/c nude mice was chosen to perform subcutaneous injection with HOS cells with or without DGKZ-knockout. Volume of tumor was checked continuously after cell implantation under overall health status for all individuals. Tumors were removed and then subjected to measure weight. Consistent with our in vitro findings, DGKZ knockdown impaired proliferation (Figures 6A,B).
Meanwhile, through bioluminescence measurement, as shown in the Figures 6C-E, the control group showed steeper progression compared with the DGKZsilenced group. After scarification, the averaged tumor weight of the control mice was extremely heavier than that in DGKZsilenced group which presented suppressed tumor growth (Figures 6F,G). These results indicated that knockout of DGKZ inhibited tumor growth of osteosarcoma in vivo.

Gene Expression Profile Analysis Revealed MYC Pathway Was a Downstream Target Affected by DGKZ
The previous data suggested that DGKZ played a key role in osteosarcoma growth. However, mechanisms underlying DGKZ-regulation are still unclear. Thus, Affymetrix GeneChip and IPA were administrated to display an overview of DGKZ possible biological interaction through DGKZ knockdown in HOS cells. Differentially expressed genes with at least a 1.5-fold change were identified. As shown in Figure 7A, 807 genes were up-regulated and 1,110 genes were down-regulated in DGKZ knockdown HOS cells compared with negative control cells. Ingenuity Pathway Analysis (IPA) revealed that several functional classifications were significantly enriched, including cell assembly/organization, cell death/survival, organismal survival, cell cycle, cellular function/maintenance, cellular development, cellular growth/proliferation, cellular movement,  tumor morphology, and cell signaling (Supplemental Figure 1). Further, IPA in "canonical pathway" module revealed that several critical pathways involved in cancer development and apoptosis such as "ILK signaling, " "signaling by Rho Family GTPases, " and "BMP signaling pathway" were activated while pathways such as "RhoGDI signaling" was inhibited by DGKZ knockdown (Supplemental Figure 2). These data demonstrated that the effects of DGKZ in osteosarcoma progression were likely dependent on regulation of several key cellular functions.
Additionally, selected gene lists obtained from the microarray analyses were uploaded to IPA system and a core biologic pathway analysis was performed to identify molecular networks. Ingenuity Pathway Analysis (IPA) revealed that after DGKZ knockdown, target genes expression in MYC pathway were altered (Supplemental Figure 3), including CCND1, CDKN2B, CDK6, MYC, PCNA, and EGR1, which were further confirmed by Western blot (Figure 7B). Taken together, these data imply that role of DGKZ in osteosarcoma pathogenesis may function via regulation of MYC signal pathway.

Identification of the ERK1/2 as DGKZ Interacting Proteins in MNNG/HOS Cells
Co-immunoprecipitated proteins were separated using SDS-PAGE and stained ( Figure 7C). Coomassie staining of the gels loaded with DGKZ-IP identified several bands that were not present in vector or IgG control. Immunoprecipitated proteins were gel extracted, trypsin digested, and identified by LC-MS/MS analysis ( Figure 7D). We identified 100 unique proteins with a protein False Discovery Rate equal or lower than 1%. We then applied a manual thresholding approach and a probabilistic PPI prediction algorithm to compute the most likely associations between each of these 100 proteins and DGKZ. Followed by co-immunoprecipitation and immunoblotting, we identified ERK1/2, a key component in Ras/Raf/MAPK pathway and a key MYC-interactor, as a candidate protein interacted with DGKZ in HOS cells (Figure 7E).

DISCUSSION
Osteosarcoma is the most common type of primary malignant bone tumor affecting both children and adults (35). Even the outcome for patients with osteosarcoma has improved dramatically by therapeutic strategies, those who suffered from distant metastasis or local recurrence continue to pose a particularly tough challenge (3). Thus, a better understanding of the molecular biology of OS is needed and may improve therapeutic efficiency. Diacylglycerol kinase zeta (DGKZ) has proven to be involved in several physiological or pathophysiological signaling pathways, including certain human carcinogenesis. In our study, we speculated that DGKZ acts as an oncogene in proliferation of osteosarcoma. Expression of DGKZ was significantly up-regulated in OS tumor samples compared with normal bone tissue, and was associated with poor prognosis. Moreover, in vitro studies, through a constructed lentivirus expressing DGKZ-specific shRNA, we further demonstrated the key role of DGKZ in cell growth and apoptosis in Saos-2/U2OS/HOS cells. Additionally, considering that a suitable animal model was needed to investigate the effect of DGKZ in vivo, we chose BALB/c nude mice, one of the most widely used models, to establish xenograft model induced by subcutaneous injection of HOS cells. Consistent with findings in vitro, our data also strongly supported the positive correlation between DGKZ expression and osteosarcoma growth. Taken together, these data indicated substantial evidence that DGKZ is a prognostic marker and potential new target for cancer therapy in osteosarcoma.
Previous data demonstrated that DGKZ was involved in tumor initiation and progression (26)(27)(28). Cai et al. (27) found that high DGKZ expression could facilitate Rho GTPase activation and promote motility of metastatic function in colon cancer. Diao et al. (28) demonstrated positive correlation between DGKZ expression and gliomagrade in glioma tumor samples. Furthermore, DGKZ knockdown in human glioma suppressed cell proliferation, hampered colony formation ability, and promoted cell cycle arrest and apoptosis. Torres-Ayuso et al. (26) explored the potential molecular mechanism of DGKZ's role in rapamycin-resistant colon cancer cell line, and found that DGKZ manipulated mTORC1 and lipogenic metabolism in colon cancer cells through SREBP-1. In line with these data, we also proved that role of DGKZ acts an oncogene in proliferation of osteosarcoma.
The regulation of DGKZ on downstream signal pathway in cancer cells is still not well-elucidated. To investigate the potential mechanism about DGKZ in regulating tumorigenesis of osteosarcoma, we conducted microarray analysis to explore alteration of cancer-related genes between normal osteosarcoma cells and DGKZ knockdown cells. Microarray data demonstrated that DGKZ's oncogene role is the result of regulating a number of different signaling pathways, which is consistent with a biofunctional role for DGKZ reported in previous study (16,(18)(19)(20)(21)(22)(23)(24)(25). IPA analysis with confirmed western blot data demonstrated that DGKZ siRNA-expressing cells posed a downregulated activity of MYC signal pathway and an altered expression of several target genes in MYC pathway. MYC proto-oncogene promotes oncogenic transcriptional amplification program in cancers and represents as an important therapeutic target for cancer therapy. In addition, misregulated expressions of MYC are often associated with osteosarcoma oncogenesis and progression (36). Taken together, it could be concluded that DGKZ might exert an enhancing effect on activity of MYC signal pathway to impact on osteosarcoma proliferation.
The MYC signaling pathway consists of a family of upstream and downstream targets that are associated with cell proliferation and growth. Whether regulation of DGKZ on downstream MYC pathway in osteosarcoma is through a direct or indirect route is still not clear. In our study, through IP-MS analysis, we provided potential evidence that the interaction of ERK1/2 and DGKZ was closely linked to MYC-dependent osteosarcoma development. ERK1/2 are widely expressed in various cells including cardiomyocytes, neurons, and hepatocytes. They are directly activated by phosphorylation of mitogen-activated protein kinase (MAPK) to promote cellular differentiation, proliferation, and survival (37). In pathological situation, such enhanced/constitutive activation of ERK1/2 existed in various human malignancies, including osteosarcoma (38)(39)(40). Various articles have showed activation of the ERK-pathway prolonged the half-life of the MYC protein and thus enhances the accumulation of MYC activity, both in normal cell growth control and oncogenesis (41,42). This finding enriched our knowledge on how DGKZ exerted an impact in osteosarcoma MYC-dependent proliferation. Thus, we speculated that DGKZ might regulate MYC signal pathway via possible interaction with ERK1/2 in carcinogenesis of osteosarcoma and utilization of DGKZ-ERK1/2-MYC interacting axis inhibitor might be an effective approach in treatment of osteosarcoma. However, the detailed mechanism of MYC pathway regulation induced by DGKZ-ERK1/2 needs further investigation.
In conclusion, our study demonstrated that DGKZ, a potential prognostic and predictive indicator in osteosarcoma, play a key role in proliferation of osteosarcoma, via its possible interaction with ERK1/2 and regulation of MYC pathway. These data provide an important starting point for future studies investigating the potential of DGKZ-ERK1/2-MYC axis as a potential new target for cancer therapy in osteosarcoma.

AUTHOR CONTRIBUTIONS
WY drafted the manuscript, participated in the design of the study, data collection. and analysis. LT and FL participated in data collection and analysis. YY was involved in drafting the manuscript. ZS designed the study and helped to draft the manuscript. All authors read and approved the final manuscript.


s2fieldsofstudy: Biology Medicine

text:
PD-1/PD-L1 Axis, Rather Than High-Mobility Group Alarmins or CD8+ Tumor-Infiltrating Lymphocytes, Is Associated With Survival in Head and Neck Squamous Cell Carcinoma Patients Who Received Surgical Resection

In current studies, the influence of tumor immune microenvironment on tumorigenesis and tumor progression has been widely explored. In the present study, we investigated the expression and significance of high mobility group box 1 (HMGB1), HMG nucleosome-binding protein 1 (HMGN1), the receptor programmed cell death 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) in head and neck squamous cell carcinoma (HNSCC). We explored whether HMGB1 and HMGN1 take part in recruiting T cells to HNSCC microenvironment. Furthermore, we assessed the prognostic value of HMG proteins, TILs, and PD-1/PD-L1 in postoperative patients. Tumor tissue sections were collected from 81 cases of patients with resectable HNSCC. All patients' information was integrated with clinical and pathological records, as well as follow-up data. We used immunohistochemistry to examine the subcellular localization and expression levels of HMGB1 and HMGN1, as well as tumor CD3+, CD8+, FOXP3+ lymphocyte infiltration, and the expression of immune inhibiting molecules PD-1/PD-L1. Results showed that there was no significant difference in the number of CD8+ and FOXP3+ T cells between the two groups with or without HMGB1 cytoplasmic expression in tumor tissues. The number of CD3+ T cells in HMGB1 cytoplasmic expression group (339.39 ± 230.76) was more than that in group without HMGB1 cytoplasmic expression (233.30 ± 230.91, P < 0.05). The number of CD3+, CD8+, and FOXP3+ T cells in HMGN1 cytoplasmic expression group [400.74 ± 224.04, 158.10 ± 112.10, 36.00(15.00, 69.00)] was more than that in group without HMGN1-cytoplasmic expression [222.84 ± 217.78, P < 0.01; 105.10 ± 108.25, P < 0.05; 13.00(6.75, 32.25), P < 0.01]. The positive rates of PD-1 and PD-L1 in tumor tissues were 29.6 and 67.9%, respectively. Multivariate analysis suggested that tumor expression of PD-L1 was an independent prognostic factor and PD-L1 overexpression indicated a poor overall survival (OS) and disease-free survival (DFS). Taken together, we concluded that HMGB1 and HMGN1 secreted by cancer cells may relate to recruitment of tumor infiltrating lymphocytes (TILs) in HNSCC. PD-1/PD-L1 axis, rather than HMG proteins or CD8+ tumor-infiltrating lymphocytes, has a critical role in tumor immune microenvironment and could predict the outcome of HNSCC patients who received surgical resection.

In current studies, the influence of tumor immune microenvironment on tumorigenesis and tumor progression has been widely explored. In the present study, we investigated the expression and significance of high mobility group box 1 (HMGB1), HMG nucleosome-binding protein 1 (HMGN1), the receptor programmed cell death 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) in head and neck squamous cell carcinoma (HNSCC). We explored whether HMGB1 and HMGN1 take part in recruiting T cells to HNSCC microenvironment. Furthermore, we assessed the prognostic value of HMG proteins, TILs, and PD-1/PD-L1 in postoperative patients. Tumor tissue sections were collected from 81 cases of patients with resectable HNSCC. All patients' information was integrated with clinical and pathological records, as well as follow-up data. We used immunohistochemistry to examine the subcellular localization and expression levels of HMGB1 and HMGN1, as well as tumor CD3+, CD8+, FOXP3+ lymphocyte infiltration, and the expression of immune inhibiting molecules PD-1/PD-L1. Results showed that there was no significant difference in the number of CD8+ and FOXP3+ T cells between the two groups with or without HMGB1 cytoplasmic expression in tumor tissues. The number of CD3+ T cells in HMGB1 cytoplasmic expression group (339.39 ± 230.76) was more than that in group without HMGB1 cytoplasmic expression (233.30 ± 230.91, P < 0.05). The number of CD3+, CD8+, and FOXP3+ T cells in HMGN1 cytoplasmic expression group [400.74 ± 224.04, 158.10 ± 112.10, 36.00(15.00, 69.00)] was more than that in group without HMGN1-cytoplasmic expression [222.84 ± 217.78, P < 0.01; 105.10 ± 108.25, P < 0.05; 13.00(6.75, 32.25), P < 0.01]. The positive rates of PD-1 and PD-L1 in tumor tissues were 29.6 and 67.9%, respectively. Multivariate analysis suggested that tumor expression of PD-L1 was an independent prognostic factor and PD-L1 overexpression indicated a poor overall survival (OS) and disease-free survival

INTRODUCTION
Head and neck squamous cell carcinoma (HNSCC) is the most frequent malignancy in the head and neck region, with about 650,000 new cases reported annually worldwide. The epidemiology of HNSCC has changed dramatically in recent decades (1). The incidence of oral cavity and laryngeal squamous cell cancers has declined, while the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has increased. Chronic infection of high-risk human papillomavirus (HPV) has been considered as one of critical factors of most HNSCCs. The prevalence of HPV in HNSCC is 60% (2), and rate is steadily increasing. In general, HNSCC is divided into two subclasses: HPV positive (HPV+) and HPV negative (HPV-). In addition, studies have demonstrated p16 was a reliable surrogate for HPV tumor status only in the OP but not in non-OP HNSCCs (3). Despite the rapid development of diagnostic and therapeutic approaches in recent years, the 5-years survival rate of patients suffering HNSCC is still <50%, which remain unchanged over the past three decades. Due to intensive research on cancer immunology and tumor molecular biology, cancer immunotherapy has shown great promise for the HNSCC therapy development.
As a complex disease, cancer involves interactions between the tumor and the immune system. Immune cells in the tumor microenvironment play an important role in tumor-associated immune responses, which may regulate tumor progression and determine the prognosis. However, immune cells infiltrating tumors are highly heterogeneous. Most of the components of myeloid and lymphoid compartments are major representatives, such as Th1 and cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, B cells, macrophages. Among all immune cells, T cell immune infiltration is the most significant predictor of patient survival. CD3 is a common differentiation antigen of mature T lymphocytes, expressed on the surface of mature T cells. CD8 + cytotoxic T cells are the main antitumor effector cells that kill tumor cells by releasing perforin or promoting apoptosis. Forkfead box P3 (FOXP3)+ Treg cells play the role of inhibiting anti-tumor immune response. Thus, in tumor-associated immune responses, the function of TILs is a combined effect regulated by interactions between different subsets. The prognostic and predictive significance of tumorinfiltrating lymphocytes (TILs) has been established for a range of tumors (4, 5).
High-mobility group (HMG) proteins are the most abundant non-histone chromosomal proteins that bind nucleosomes and regulate chromosome architecture and gene transcription.
HMG proteins can be classified into HMGA, HMGB, and HMGN families according to unique structural motifs. On the other hand, HMG proteins in the extracellular milieu, which are released from injured, dying or stressed cells, have also been recognized as danger-associated molecular pattern (DAMP) molecules based on their shared chemical property of hydrophobicity and function of stimulating the immune system (6). As "alarmins, " HMGB1 and HMGN1 are shown to exhibit similar chemoattracting and activating effects on dendritic cells (DCs), as well as the capacity to enhance innate and adaptive immune responses. A study found that high cytoplasmic expression of HMG proteins correlated with high levels of TILs in HER2-positive breast cancers (7). However, the expression patterns of HMG proteins in HNSCC tissues are largely unknown.
Although some studies confirm the association between TILs and survival in HNSCC patients, the prognostic role of TILs is still controversial. One reason might be that PD-1/PD-L1 axis plays a role in silencing immune surveillance by rendering CD8+ cytotoxic T cells anergic. In the present study, we assessed the subcellular localization and levels of HMGB1 and HMGN1 expression. We also further evaluated the association of HMG proteins and TILs in HNSCC tissues and explored their correlation with clinicopathological variables, including patient survival. In addition, we also analyzed the predicting value of PD-1/PD-L1 status on postoperative outcome.

Patients and Tissue Specimens
A total of 81 HNSCC patients who underwent surgery from Department of Head and Neck Oncology, Tianjin Medical University Cancer Institute and Hospital (TMUCIH), Tianjin, China, between August 2009 and October 2013 were enrolled. All patients had formalin-fixed, paraffin-embedded tissue samples available for analysis and were diagnosed as HNSCC pathologically for the first time, within the enrollment time. The TNM staging system was used to classify the tumors in accordance with the American Joint Committee on Cancer classification. The Institutional Review Board approved the protocol for this study, and written informed consent was obtained from each patient before enrollment.
All patients were followed up until December 2017. The definition of overall survival (OS) was the period from the surgery to death of any reason or to the time of last follow-up. disease-free survival (DFS) was defined as the period after curative treatment (surgery) to the time when recurrence was diagnosed or to the last date of follow-up when no disease can be detected. Patients lost to follow-up were censored as of the last day of follow-up. Patients who died from causes other than HNSCC were censored at the date of death.
Tumor sections were accessed by a consensus of two pathologists who did not know the characteristics of the tissue. Five images were randomly taken at high magnification on each slide. Positive HMGB1 and HMGN1 expression existed in nucleus of tumor cells, mesenchymal cells, as well as immune cells. Based on the staining intensity of nucleus in tumor cells, tumor tissues were divided into high expression group and low expression group. According to whether HMGB1 or HMGN1 expressed in the cytoplasm of tumor cells, the tissue sections were divided into cytoplasmic positive group and cytoplasmic negative group. In the case of CD3+, CD8+, and FOXP3+ staining, the positive cells per core were counted manually and average counts of five fields were used for analysis. Positive CD3+, CD8+, or FOXP3+ expression represent the number of total T cells, CTLs or Tregs in the tumor microenvironment, respectively.
In the 81 cases of HNSCC, tissue sections were considered as PD-1 positive when at least 10% of lymphocytes in tumor microenvironment showed PD-1 staining. PD-L1 positive were defined as tumor cells having membranous or cytoplasmic staining. According to previous studies, <5% tumor cells staining were defined as negative (-, scored as 0). For samples with PD-L1 positive ≥5%, based on the staining intensity from light brown to general brown and dark brown, further divided the expression levels into three degrees: weakly (+, recorded as 1), moderately (++, recorded as 2), and strongly positive (+ + +, recorded as 3) correspondingly. Negative and weak positive expression was considered as low expression while moderately and strongly positive expression was described as high expression.

Statistical Analysis
All statistical analyses were performed using Statistical Package for the Social Sciences version 17.0 (IBM Corp., USA), and the normality of the data was evaluated using the Kolmogorov-Smirnov test. Given that there was no clinically defined cutoff points for the number of TILs in tumor microenvironment, the median numbers of CD3+, CD8+, and FOXP3+ T cells (227 for CD3+, 90 for CD8+, and 18 for FOXP3+) were used as a cutoff point to define the TILs-low and TILs-high groups. Person chi-square (χ2) test or Fisher's exact test were used to compare categorical variables. The correlations between HMGB1 or HMGN1 expression and TILs were assessed using Spearman's correlation analysis. Kaplan-Meier analysis was the method used to calculate the overall survival (OS) and disease-free survival (DFS), then the OS and DFS was compared by log-rank test. Multivariable analysis of the independent factors which were correlated with survival was performed by the Cox proportional hazard model. Statistically significant is considered as the twotailed P < 0.05.

Patient Characteristics and Baseline Clinicopathologic Characteristics
This analysis included 81 HNSCCs, including cancers of the oropharynx (n = 17), oral cavity/lip (n = 38), larynx (n = 17), and hypopharynx (n = 9). Patients were predominantly male (80.2%) and the majority of tumor tissue samples were moderately (58.0%) differentiated. Median age was 56 years (interquartile range, 49-64 years), and most patients had a history of ever tobacco and alcohol use.

Expression of HMG Proteins in Patients With HNSCC and its Relationship With TILs
Epithelial cells and lymphocytes showed nuclear expression of HMGB1 and HMGN1. However, various immunostaining patterns of these two proteins could be observed in carcinoma cells (Figure 1). The correlation of HMG alarmins expression with clinicopathological characteristics were shown in Tables 1 and 2. Positive cytoplasmic expression of HMGB1 was significantly associated with a low lymph node positive rate (P = 0.004) and p16 negativity (P = 0.049). Besides, nuclear expression of HMGB1 was correlated with drinking status (P = 0.040), while nuclear expression of HMGN1 was associated with p16 status (P = 0.033). The nuclear expression of HMGB1 and HMGN1, and the cytoplasmic expression of both proteins were significantly correlated with each other (rho = 0.456, P < 0.001 for nuclear expression; rho = 0.518, P < 0.001 for cytoplasmic expression). The correlation between cancer tissue expressed HMGB1/HMGN1 and stromal immune cell infiltrate were then observed. The correlation of TILs with clinicopathological characteristics were shown in Table 3. High CD8+ T cell count was significantly associated with drinking. As shown in Table 4, nuclear expression of HMGB1 was inversely correlated with CD3+ T cells, CD8+ CTLs, and FOXP3+ Tregs, while cytoplasmic expression of HMGN1 was significantly associated with these three groups of TILs. Further analysis

HMG Alarmins, Tumor-Infiltrating Lymphocytes, PD-1/PD-L1 Status and Survival
The levels of cancer expressed PD-1 and PD-L1 were also detected by IHC. PD-1 was considered as negative or positive according to the staining proportion, and PD-L1 was recorded as 0, 1, 2, and 3, respectively representing negative, weakly positive, moderately positive and strongly positive, in accord with both the intensity and proportion of staining (Figure 2). The positive rates of PD-1 and PD-L1 in tumor tissues were 29.6 and 67.9%, respectively. Next, we investigated the role of HMG alarmins expression, TILs pattern, and PD-1/PD-L1 status on patients' survival. Optimal cut-offs for low vs. high staining were made as described in the statistical methods. Patients with negative HMGB1 or HMGN1 cytoplasmic expression seemed like to show an improved effect on OS and DFS, even though there was no statistical significance (Figure 3). For TILs, low FOXP3+ regulatory T cell count but not CD3+ T cell count and CD8+ T cell count was suggested to have a favorable influence on OS and DFS (Figure 4). The median follow-up duration was 41 months (ranging from 2 to 98 months). At the end of the follow-up period, there were 48 deaths and 33 survivals. Patients' 1-, 3-, and 5-years OS rate were 88, 51, and 21%, respectively, and the corresponding DFS rate was 70, 44, and 17%, respectively. Univariate analysis showed that OS has significant correlation with TNM stage (P = 0.001), tumor size (P = 0.014), lymph node metastasis (P = 0.000), Treg count (P = 0.009), and PD-L1 expression (P = 0.007), while DFS was directly influenced by histology (P = 0.007), TNM stage (P = 0.001), tumor size (P = 0.010), lymph node metastasis (P = 0.002), Treg count (P = 0.038), and PD-L1 expression (P = 0.002). Multivariate analysis showed lymph node metastasis, Treg count and PD-L1 expression on tumor cells were independent prognostic factors for OS, while TNM stage, Treg count and PD-L1 expression could be regarded as independent prognostic factors of DFS (all P < 0.05, Tables 5, 6). Patients with low PD-L1 expression on tumor cells had a more favorable outcome.

DISCUSSION
Traditional therapeutic methods have unsatisfactory curative effects on HNSCC. Despite improvement in operation techniques and targeted therapy for immune checkpoints, the prognosis of head and neck cancer patients remains poor. Therefore, there is an urgent need to explore the key mediators and the underlying molecular mechanisms associated with tumor growth, recurrence and metastasis after curative treatment like surgical resection. Recently, accumulated research suggests that inflammatory mediators may play a role either in tumor establishment or in progression. In other words, the tumor microenvironment can be described by the chronic inflammation process.
HMGB1 and HMGN1 are recently identified alarmins which are a subset of damage-associated molecular patterns capable of inducing both the recruitment and activation of dendritic cells (DCs) (8,9). Then DCs can promote specific immune responses against many tumor-associated antigens. Particularly, HMGN1 has been shown to be critical for the generation of anti-tumor immunity against mouse thymoma and melanoma (10). In our study, we demonstrated that cytoplasmic expression of HMGB1 in tumor tissue was associated with CD3+ T lymphocyte count but were not correlated with the count of FOXP3+ T cells, while HMGN1 in cytoplasm was correlated with CD3+ T cell count, CD8+ T cell count and FOXP3+ regulatory T cell count, suggesting that the secretion of these two HMG alarmins in tumor tissue may contribute to the influx of TILs.
Although our results demonstrated that secretion of HMG alarmins by tumor cells could recruit CD3+ and CD8+ T cells into tumor microenvironment, further analysis showed that the count of CD3+ and CD8+ T cells were not directly associated with postoperative survival. Meanwhile, the prognostic significance of HMG alarmins expression in HNSCC tissues has not yet been proven. However, our present work indicated that tumors with high FOXP3+ Tregs infiltration tended to have a poorer prognosis. This is in accordance with the previous studies which indicated that regulatory T cells played direct roles in promoting immune evasion and were correlated with poor prognosis in cancer (11). The infiltrated Treg cells in tumors, as one of the major immunosuppressive cellular population in the tumor microenvironment, accelerate the progression of malignant tumors by promoting tumor growth, enhancing tumor-associated angiogenesis, inhibiting adaptive immunity, and helping tumor cells invade surrounding normal tissues and metastasize to distant organs. Recently, accumulating studies have verified the correlation between TILs and survival in several kinds of tumors. However, the prognostic role of TILs is still controversial. Some TIL subsets, especially CD8+ TILs, could be regarded as valuable prognostic factors in HNSCC (12). In our present study, though TILs infiltration is associated with HMG proteins, no statistical correlation with postoperative survival was observed. It may be ascribed to the expression of immune inhibitory molecules. Then, we wondered how the relationship of each molecule to survival related to tumor lymphocyte infiltration. The checkpoints of PD-1 and PD-L1 have crucial roles in the formation of "immune privilege" regions, tumor development, and immune evasion. PD-L1 is expressed in numerous tumors, including HNSCC. Binding of PD-L1 to its receptor PD-1, on activated T cells, suppresses T-cell responses (13). Our present work showed that high tumor expression of PD-L1 was an independent predictor of poor HNSCC-specific survival. Similar to our results, high expression of PD-L1 was found to be associated with poor survival in melanoma (14), non-small cell lung cancer (15), colorectal (16), and renal cell cancer (17). On the other hand, other studies in melanoma (18), gastrointestinal-stromal tumors (19), and colorectal cancer (20) have shown the opposite. Lately, a meta-analysis suggested the correlation between PD-L1 within tumors and the prognosis may vary among different tumor types and by the type of tissue sampling used for assessment. Therefore, the expression of PD-L1 on tumor cells is well-known but in order to assess its function in the (anti-) tumor immune response and its association with PD-1 expression on tumor infiltrating immune cells, further investigation is needed. As cancer molecular biology and immunology developed rapidly, targeted therapy for PD-1/PD-L1 axis has shown enormous development prospects for HNSCC treatment. In 2016, the results of two milestone trials ]KEYNOTE-012 (21,22) and CheckMate 141 (23)] promoted the antagonists of PD-1 / PD-L1 axis such as pembrolizumab (MK-3475) and nivolumab (BMS-936558) which become the earliest targeted therapeutic drugs for HNSCC approved by the US Food and Drug Administration (FDA). At present, the two drugs have already been the fourth standard therapeutic method after surgery, radiotherapy, and chemotherapy for HNSCC.
In the current study, statistical analysis showed that the number of CD3+ T cells in HMGB1 cytoplasmic expression group was more than that in group without HMGB1 cytoplasmic expression, while both CD3+ and CD8+ T cell counts in HMGN1 cytoplasmic expression group were more than that in group without HMGN1 expression. Although HMGB1 and HMGN1 function both as chromatin binding proteins and as alarmins, they are otherwise completely distinct proteins and have some distinct roles. When released by tumor cells, extracellular HMGN1 function as a Th1-polarizing alarmin that attracts CD8+ CTLs and promotes the induction of adaptive antitumor immune responses. However, the upregulated expression of PD-L1 on tumor cells could inhibit the antitumor activity of CD8+ CTLs. HNSCC patients with cytoplasmic expression of HMGN1 and high tumoral PD-L1 expression, which suggests adaptive immune resistance, may benefit from PD-L1 or PD-1 blockade. But our results showed that there was no significant difference in the number of CD8+ CTLs between the two groups with or without HMGB1 cytoplasmic expression in tumor tissues. Therefore, in our opinion, compared to patients with HMGB1 cytoplasmic expression, HNSCC patients with both HMGN1 cytoplasmic expression and high expression of PD-L1 would be expected to get more benefit from anti-PD-L1/PD-1 therapy. In our present work, we found that 31 out of 81 patients had positive cytoplasmic expression of HMGN1 in tumor tissue. Among these 31 patients, 20 patients' tumor tissue expressed PD-1 on lymphocytes, and 24 patients' tumor tissue highly expressed PD-L1. Taken together, there were totally 16 patients whose tumor tissues were harboring HMGN1 cytoplasmic expressed and PD-L1 positive tumor cells, as well as PD-1 positive tumor infiltrating lymphocytes (TILs). We assumed that HNSCC patients with this type of molecular feature may benefit the most from PD-1/PD-L1 blockade.
In our opinion, the present work has several strong characteristics. We have examined the expression of HMG proteins and immune inhibitory molecule PD-L1 in tumor, as well as surrounding TILs. Our study is first to examine the coexpression of HMG proteins and PD-1/PD-L1 in HNSCC. There are of course also limitations in our study. Firstly, we investigated the expression of HMG proteins, TILs infiltration and PD-1/PD-L1 status only in patients who have a chance to undergo surgery, the data were not available for patients with advanced HNSCC and further study is needed. Secondly, the consensus to determine positive PD-1/PD-L1 marker threshold on biopsy tissue specimens by IHC is still a challenge. Substitutive tests such as RNA-based assays can be used to demonstrate the effects of PD-1/PD-L1 status in prognostic significance of HNSCC. Further studies are required to confirm the importance of HMG alarmins in tumor microenvironment.
In conclusion, our study found that the release of highmobility group alarmins in tumor tissues of HNSCC patients was correlated with the count of TILs, in other words, it might involve in cellular immunity. However, further analysis suggested that PD-1/PD-L1 axis, rather than HMG alarmins or CD8+ tumorinfiltrating lymphocytes, associated with survival. Our results may pave a way for evolution of immunotherapy of head and neck cancer.

ETHICS STATEMENT
The study was based on the recommendations from biomedical research guidelines involving human participants designed by the National Health and Family Planning Commission of China. The study is approved by the Ethical Committee of TMUCIH, and we obtained written informed consent from each subject on the basis of the Declaration of Helsinki.

AUTHOR CONTRIBUTIONS
XR, FW, and FY designed the study protocol. FY, ZZ, and JL performed the experiments. FY, YZ, and FW analyzed the collected data. FY wrote the manuscript. XR and FW revised the manuscript.

FUNDING
This work was partially funded by a grant from the National Natural Science Foundation of China (No. 81672697).


extfieldsofstudy: Medicine Biology

text:
Characterization of the complete mitochondrial genome of cape elephant shrew, Elephantulus edwardii

Abstract In this study, the complete mitochondrial genome of cape elephant shrew, Elephantulus edwardii, was determined through sequencing of PCR fragments. The complete mitochondrial genome of E. edwardii was 16,552 bp in length and and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 32.7% A, 29.5% T, 25.0% C, and 12.9% G, with a total G+C content of 37.9%. By phylogenetic analysis using Bayesian method, E. edwardii showed the closest relationship with another Elephantulus speceis (Genbank accession: AB096867.1).

The cape elephant shrew, Elephantulus edwardii, is neither closely related to shrews nor to rodents (mice), but belong to the order Macroscelidea (family Macroscelididae) with currently 17 species in four genera distributed in Africa and is fundamental to evaluating its recognition as vulnerable in the IUCN Red List.
In this study, the specimen was collected from the Matsikamma mountain (18 49.518 0 E, 31 44.203 0 S), Africa. The total DNA was extracted from muscular tissue, using the commercial Animal Tissues Genomic DNA Extraction Kit (Solarbio, BJ, CN) following the manufacturer's instructions, and then used as the template for polymerase chain reaction (PCR) amplifications. Fifteen pairs of primer were designed based on mitogenome of Elephantulus sp. VB001 (Genbank accession: AB096867.1). The muscular tissue was stored at À80 C and extracted genomic DNA was sotred at À20 C in the Zoology comprehensive laboratory from College of Animal Science and Veterinary Medicine. The isolated DNA was sent to sequencing company (BGI Tech, Shenzhen, China). The amplified products were sequenced using the amplification primers. All sequencing was done by a commercial sequencing service (BGI Tech, Shenzhen, China). Some small number of PCR products that could process complex secondary structures or high AþT content, were cloned into the PMD-19T vector (TaKaRa, Dalian), then transformed to JM109 competent cell (TaKaRa, Dalian) for sequencing. All sequencing sequences were assembled with the program Seqman in the DNASTAR package (Burland 1999).
Protein coding genes were predicted using ORF Marker tool in UGENE (Okonechnikov et al. 2012) under the vertebrate mitochondrial genetic code. Protein-coding regions and ribosomal RNA genes were also annotated manually and confirmed by comparison to the mitogenome of Elephantulus sp. (Genbank accession: AB096867.1) that available in Genbank. The transfer RNA (tRNA) genes were identified and assigned putative secondary structures using the program tRNAscan-SE (Lowe et al. 1997) or by manually identifying potential secondary structures and anticodon sequences through visual inspection. The graphical map of the complete mitochondrial genome was drawn using the online sofware OrganellarGenomeDRAW (Lohse et al. 2007).
For phylogenetic analysis assessing the relationship of this mitogenome, we selected other 33 Eutheria mitogenomes downloaded from GenBank. The genome-wide alignment of all mt genomes was done by HomBlocks (Bi et al. 2018), resulting in 20,840 characters in total, including almost all whole or partial PCGs and rRNA genes. The whole genome alignment was analyzed by PhyloBayes ver. 3.3 (Lartillot et al. 2009) under the GTRþG model. Four independent MCMC analyses were run for 10,000 cycles in PhyloBayes. Convergence was checked based on time-series plots of the likelihood scores using Tracer (http://tree.bio.ed.ac.uk/software/tracer/). The first 25% cycles were discarded as burn-in, and the remaining trees were summarized to obtain Bayesian posterior probabilities (BPPs). The resulting tree was represented and edited using FigTree v1.4.1 (http://www.umiacs. umd.edu/$morariu/figtree/). As shown in Figure. 1, the phylogenetic positions of these 34 mt genomes were successfully resolved with full BPPs supports across almost all nodes. As expected, Elephantulus edwardii showed the closest relationship with Elephantulus sp. VB001. And they clustered as Macroscelididae clade with Macroscelides proboscideus.

Disclosure statement
No potential conflict of interest was reported by the authors. Figure 1. Phylogenetic relationships among 34 Eutheria mt genomes. This tree was drawn without setting of an outgroup. Nodes exhibit 100% posterior probability (PP) were labelled by green points.


extfieldsofstudy: Chemistry Medicine

text:
Comparison of the effects of progesterone and 17 β-estradiol on Schwann cell markers expression in rat adipose-derived stem cells

Steroids promote the myelination and regeneration in the peripheral nervous system. Whereas, little is known about the inducing effects by which the hormones exert their effects on Schwann cells differentiation. This could be revealed by the expression of Schwann cell markers in adipose-derived stem cells (ADSCs). The purpose of this study was to present the effects of progesterone and 17 β-estradiol on the Schwann cell markers in rat ADSCs. The mesenchymal stem cell markers (CD73, and CD90) were assayed by flow cytometry. Rat ADSCs were sequentially treated with β-mercaptoethanol, and all-trans-retinoic acid, followed by a mixture of basic fibrobroblast growth factor, platelet-derived growth factor, forskolin and heregulin. In experimental groups, forskolin and heregulin were substituted by progesterone and 17 β-estradiol. After induction, the expression of Schwann cell markers P0, and S-100 and the cellular immunocytochemical staining positive rate of anti-S100 and anti-glial fibrillary acidic protein (GFAP) antibodies were compared in the experimental and control groups. Progesterone and 17 β-estradiol triggered P0 and S-100 genes expression and induced a cellular immunocytochemical staining positive rate of S-100 and GFAP in rats ADSCs. Progesterone induced these changes stronger than 17 β-estradiol. Thus, progesterone may induce rat ADSCs toward Schwann-like cells by expression of Schwann cell markers and is more potent than 17 β-estradiol in the expression of these markers.

Introduction
Schwann cell is a principle glia and plays a pivotal role in the development, regeneration and maintaining of the peripheral nervous system. 1,2 Schwann cells release neurotrophic factors and involve in peripheral axons myelination, directional guidance of neurons, and cellular fragments removal. 2 Various studies have shown that implanted Schwann cells in nerve guidance channels improve the advantageous endogenous responses to nerve injury and allow nerve regeneration. [3][4][5] However, the clinical application of Schwann cells is debatable because it is complicated to find enough numbers of Schwann cells.
Adipose-derived stem cells (ADSCs) are useful in regenerative medicine. 6 Several recent studies have introduced ADSCs as a source of multipotent stem cells which can be differentiated into several cell lines. 7,8 Obtaining of ADSCs is easy, and efficient; it shows very low donor site morbidity. 9 In addition, adipose tissue contains more than a thousand-fold more mesenchymal stem cell (MSC) compared to bone marrow. 10,11 The MSCs and glial cells are targets for progesterone and estrogen. [12][13][14][15] Schwann cells synthesize and metabolize neurosteriods such as progesterone and 17 βestradiol and express steroid receptors. 16 Neurosteroids regulate the expression of myelin proteins and transcription factors with a key role in Schwann cells physiology and in their myelinating program. 16,17 Progesterone plays a significant function in peripheral nerves myelination. 17,18 It could improve the formation of new myelin sheaths subsequent to the mouse sciatic nerve damage. 19 In addition, progesterone has promoted axons myelination when added to explanted cultures of rat dorsal root ganglia, 12,19 accelerated the time of myelination nitiation and increased the amount of myelin synthesis in co-cultures of Schwann cells and sensory neurons. 20 Estrogens regulate re-myelination and promote neuronal survival via expression of growth factors by glial cells. 21,22 Interestingly, previous study has indicated that progesterone stimulates the expression of Schwann cell markers in bone marrow MSCs, 13 but the stimulating effect of neuroesteroids on the expression of Schwann cell markers in ADSCs has not been investigated. In the current study, a comparative study of the inducing effects of progesterone and 17 β-estradiol with a focus on the expression of Schwann cell markers in the ADSCs culture was carried out.

Materials and Methods
Isolation and culture of ADSCs. The ADSCs were isolated from adult 4-6 weeks old male Wistar rats (n =6). Animal experimental procedures were approved by the Research Committee of Kashan University of Medical Sciences (N:5219, 2010). The rats were euthanized and fat pads were removed carefully from inguinal region. The fat pieces were washed with phosphate buffered saline (PBS) and mechanically dissociated using sterile scalpel blades. After removal of debris, the adipose tissue was enzymatically digested for 60 min at 37.00 ˚C using 0.50 mg mL -1 collagenase type I (Sigma-Aldrich, Saint Louis USA). The solution was centrifuged to separate the floating population of mature adipocytes from the pelleted cells of stromal vascular fractions. The suspension containing dissociated tissue cells was neutralized by the addition of ADSC medium containing Dulbecco's modified Eagle medium (DMEM/F12; Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin solution and centrifuged at 1000 g for 7 min, re-suspended and filtered. The cells were centrifuged and the pellet was resuspended in ADSC medium and transferred to a T75 flask centrifuged at density of 1.00 × 10 6 cells. The culture medium was changed every other day until the cells became 80.00% confluent. The cells were incubated at 37.00 ˚C with 5.00% CO2 and passaged up to three times using trypsin-ethylene-diaminetetraacetic acid (EDTA; Gibco). 23 Cell viability of the cultured cells in each passage was frequently about 95.00%. Cell count was determined using a manual hemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany).
Characterization of ADSCs properties. At passages 3-5, cells were harvested by the addition of 0.20% trypsin-EDTA and washed by PBS 3 times. Aliquots were incubated with different rat antibodies (CD90, CD45, CD73 or VEGFR2) 8,23,24 conjugated with fluorescein isothiocyanate (Chemicon, Temecula, USA) in dark, at room temperature for 45 min. The phenotype of cultured was analyzed by flow cytometry using a FACscan cytofluorometry (Becton Dickinson, San Jose, USA).
Differentiation to a Schwann cell-like phenotype. The ADSCs medium was removed from the cultures at passage 3 and replaced with medium containing 1 mM βmercaptoethanol (Sigma) for 24 hr. Then, cells were washed and fresh medium supplemented with 35 ng mL -1 all-trans-retinoic acid (Sigma) was added. After 72 hr, cells were washed and the medium was replaced with induction medium in 6 different groups: In the first group, the cell differentiation medium supplemented with 5 ng mL -1 platelet-derived growth factor (PDGF; R&D), 10 mL -1 basic fibroblast growth factor (bFGF; Gibco), 5.00 μM forskolin (Sigma) and 200 ng mL -1 heregulin-β (Milipore) was added (positive control group); forskolin and heregulin were replaced with progesterone (10 -6 M; Sigma) respectively in the second and third groups and 17 β-estradiol (10 -6 M; Sigma ) respectively in the fourth and fifth groups. In the sixth group (negative control group), the medium contained only 5 ng mL -1 PDGF and 10 ng mL -1 bFGF. The cells cultures were followed by incubation for eight days. 13 Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Following the differentiation period, total RNA was extracted with RNX plus (Ceinna-Gen, Tehran, Iran). Complementary DNA (cDNA) synthesis was carried out from total RNA using Fermentas kit (Fermentase, Waltham, USA) based on the manufacturer's instructions. The cDNA was amplified using the primers designed for S-100 and P0 genes ( Immunocytochemistry. The ADSCs of different groups were digested by trypsin-EDTA, and seeded into 24 cell plates following centrifugation and collection. Then, the cells were incubated at 37 ˚C, washed with PBS, dried and fixed in 4.00% paraformaldehyde for 20 min. Following permeablization using 0.40% Triton X-100 and blocking by 5.00% normal goat serum, the cells were incubated with primary antibodies against S-100 (1:100; Sigma), or GFAP (1:100; Sigma) at 4 °C for 1 hr. After washing, the cells incubated with FITC-conjugated anti-rabbit IgG (1:100; Sigma) for 45 min at room temperature. The 4ʹ6ʹdiamidino-2-phenylondole dihydro-chloride (DAPI) was used to determine the nuclei as a counterstain. Finally, the cells were examined using an inverted fluorescence microscope (Eclipse Ti-S; Nikon, Tolyo, Japan) and the percentage of immuno-positive cells was calculated in five randomly selected fields at 40× magnification.
Statistical analysis. The data were analyzed using one-way ANOVA and Tukey's post-hoc test and the results were expressed as mean ± standard deviation. A p-values less than 0.05 was considered statistically significant.

Results
Adipose tissue was enzymatically digested and stromal cells were isolated. The cells were cultured and after 4 to 5 days, they proliferated rapidly and their condensation reached to approximately 80.00 to 90.00%. The cultured cells exhibited fibroblast-like morphology in microscopic observations.
Most of MSCs were negative for the expression of CD45 and VEGFR2 but they were positive for CD90 and CD73 expression. These data demonstrated the mesenchymal nature of the isolated cells.
Differentiation. The ADSCs expressed low levels of S-100, and P0 mRNAs. The ADSCs at passage three were treated with a mixture of glial growth factors containing FGF, PDGF, forskolin, and heregulin for a period of one week. This treatment increased the expression of Schwann cell markers (S-100 and P0) intensely at mRNA level. Progesterone (groups 2 and 3) or 17 βestradiol (groups 4 and 5) increased these markers compared to the negative control group (group 6). The expression of S-100 mRNA in ADSCs in the differentiation media containing progesterone and heregulin (groups of 1-3) was significantly higher than other groups (p < 0.0001), (Fig. 1).
The expression level of P0 in the presence of progesterone and heregulin was near to positive control group (Fig. 2).   In immunocytochemistry experiment, S-100 and GFAP positive cells were counted in five fields under fluorescent microscope. The undifferentiated cells of the control group had no expression of S-100 and GFAP. The staining intensity of S-100 and GFAP of cells in groups 2 to 5 (experimental groups) was more strongly positive than that of cells in group 6 (negative control group). In group 2 (in the presence of progesterone and heregulin), mean number of the S-100 and GFAP staining positive cells was 20.00 ± 5.29 and 18.30 ± 1.15, respectively. In group 3 (in the presence of progesterone and forskolin) mean number of the S-100 and GFAP staining positive cells was 21.30 ± 6.50 and 18.30 ± 1.15, respectively. In group 4 (in the presence of 17 β-estradiol and heregulin), mean number of the S-100 and GFAP staining positive cells was 20.30 ± 2.80 and 20.30 ± 1.15, respectively. In group 5 (in the presence of 17 β-estradiol and forskolin), mean number of the S-100 and GFAP staining positive cells was 25.30 ± 4.70 and 11.60 ± 3.50, respectively. Immunocytochemistry showed that the expressions of S-100 and GFAP in the experimental groups were significantly superior to those of the negative control group (group 6), (p < 0.0001). Also, the GFAP proteins were significantly expressed higher in the cytoplasm of group 4 cells than group 5 cells (p < 0.001), (Figs. 3 and 4).   Fig. 3. A, and B) Quantitative analysis of morphology and expression of S100 (I), and GFAP (II) proteins. a: Significant difference with 1 st group, (p < 0.0001); b: Significant difference with 6 th group, (p < 0.0001); c: Significant difference between 4 th and 5 th groups (p < 0.001).

Discussion
The purpose of this study was to examine the inducing effects of progesterone and 17 β-estradiol on expression of Schwann cell markers in ADSCs. According to the results of present study, treatment of ADSCs with progesterone or 17 β-estradiol could induce the expression of Schwann cell markers.
Adipose tissue contains a high number of multipotent MSCs which are able to differentiate into different cells. The ADSCs are useful for a range of therapeutic applications such as treatment of peripheral nerve injuries. [25][26][27] Several experiments have indicated that ADSCs can be differentiated into a Schwann cell phenotype as well. [28][29][30] Dezawa et al. differentiated bone marrow separated MSCs into myelinating cells with the ability to induce nerve regeneration. 31 This method is based on the effect of a variety of inducers that used one by one. 32 Our experiment showed that the sequential administration of various factors β-mercaptoethanol and retinoic acid, followed by a mixture of bFGF, PDGF, forskolin and hereguiln is more efficient than other inducer mixtures and confirmed Dezawa method. The combination of β-mercaptoethanol and retinoic acid in culture media is an appropriate pre-inducer 21 and a mixture of bFGF, PDGF, forskolin and hereguiln has a synergistic effect on Schwann cell differentiation. 31 In Schwann cell development process, forskolin can increase the level of intracellular cyclic adenosine monophosphate (cAMP) causing cells response to the trophic factors leading to myelin marker of P0 mRNA expression. 33,34 Hereguiln, a subtype of neuregulin, is one of the crucial axon-derived signals influencing Schwann cells development. 35 Neurosteriods involve in the regulation of Schwann cells proliferation and their cellular products. [36][37][38] Progestins have the ability to activate heregulin receptors of ErbB2 and ErbB3. 39 In the present study, forskolin and heregulin were replaced by progesterone and 17 βestradiol. Among the various Schwann markers, we particularly focused on P0, S-100, and GFAP. In our experiment, P0, a representation marker of myelinating cells, was expressed in ADSCs in the presence of progesterone. Melcangi et al. have showed that the expression of P0 is under the control of progesterone receptor and progesterone increases its expression in rat Schwann cell culture. 40,41 Our data support those of Roglio et al. that suggesting that progesterone may be necessary for inducing P0 synthesis. 16 Due to estrogen function in tissue and organ development through regulating cell proliferation and differentiation, we hypothesized that 17 β-estradiol induces expression of Schwann cell markers in ADSCs. Based on our results, S-100, P0, and GFAP were expressed significantly in 17 β-estradiol groups (the fourth and fifth groups). The MSCs are anabolic targets of estrogen action, via estrogen receptor α activation. 42 17 β-estradiol induces an earlier onset of axonal regeneration and myelination. 43 The estradiol alone had no effect on cell multiplication, but in the presence of forskolin it became a potent mitogen. 36 In the present study, in the fifth group, 17 β-estradiol in the presence of forskolin could induce a significantly higher expression of P0 mRNA compared to the forth group. It seems that estradiol becomes an inducer for ADSCs when levels of cAMP are elevated. 44 However, in our study, 17 β-estradiol was a weaker inducer compared to progesterone for expression of Schwann cell markers in ADSCs. Higher expression of GFAP protein positive cells in the presence of 17 β-estradiol and heregulin than combination of 17 β-estradiol and forskolin is evidence for the role of heregulin in induction of ADSCs. Dezawa et al. believed that no growth factors can be a substitute for heregulin in Schwann cell differentiation. 31 Our study showed that progesterone has an inducing effect similar to that of heregulin and forskolin in expression of Schwann cell markers. This is consistent with the study of Movaghar et al. that showing that progesterone could induce the transdifferentiation of bone marrow stem cells (BMSCs) into Schwann cell phenotype. 13 It seems that the combinations of heregulin and forskolin, 31 forskolin and progesterone 13 or heregulin and progesterone (present study) have cumulative effects in the differentiation of MSCs into Schwann-like cell phenotype. However, in contrast to Movaghar et al., the strongest expression of Schwann cell markers was not seen in groups treated with progesterone, but it was seen in group 1 treated with heregulin and forskolin. Owing to the differences in the immunophenotype, transcriptome, proteome, and immunomodulatory activity, BMSCs and ADSCs have diverse potential in differentiation into variety of cells. 6,45,46 Although it has been shown that heregulin is able to induce progesterone receptor activation 47 it seems that the combination of heregulin and progesterone induces BMSCs and ADSCs through different pathways.
In conclusion, we induced the rat ADSCs by progesterone and 17 β-estradiol in vitro and demonstrated the expression of Schwann cell markers. There are still some limitations in identification of the Schwann cell markers, hence further studies on the induction mechanism of progesterone and estradiol and differentiation of ADSCs into Schwann-like cell phenotype are recommended.