MATERIALS Dulbecco’s modified eagle medium (DMEM) supplemented with 10%

MATERIALS AND METHODS Bioinformatic analyses of has-mir-127 and REPIN1 data in gliomaThe publicly available portal LinkedOmics (www.linkedomics.org) that contains multi-omics data from the 32 TCGA Cancer types was adopted to uncover the effect of has-mir-127 and REPIN1 on the survival of glioma cells and their expression profile in different tissue types. The LinkFinder analytical module was used to search for mRNA expression signatures (from mRNAseq data) associated with has-mir-127. The Pearson correlation analysis was applied to assess the correlation of has-mir-127 with mRNA expression signatures. Analysis results were visualized by scatter plots, box plots, or Kaplan-Meier plots. To derive biological insights from the association results, the LinkInterpreter module was used to perform enrichment analysis based on Gene Ontology and biological pathways.  Cell lines and culture The normal human glial cell HEB and two glioma cell lines U87 and LN-229 were al purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, South Logan, UT, USA), 50 ug/mL streptomycin and 100 ug/mL penicillin at 37 °C in an incubator containing 5% CO2. Quantitative real-time PCR Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). An aliquot of purified RNA was reverse-transcribed Reverse into cDNA using the Transcription Kit (Takara Bio, Inc., Otsu, Japan) in accordance with the manufacturer’s instructions. The amplification of REPIN1 was performed on a Biorad Realtime PCR platform. The PCR reaction cycling conditions for the 30 cycles were: 94 °C for 2 min, 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min and 72 ? for 10 min. GAPDH was used as an endogenous control. Primer sequences were as follows: for REPIN1, forward 5 GAT-CGG-GCC-TTT-TTG-TGC-TC-3? and reverse 5?-CTT-GCG-AGT-GAG-CCA-TTT-CG-3?; for GAPDH, forward 5?-GCA-ACT-AGG-ATG-GTG-TGG-CT-3? and reverse 5?-TCC-CAT-TCC-CCA-GCT-CTC-ATA -3?. The expression level of mature hsa-mir-127-5p was determined with a TaqMan miRNA assay kit (Applied Biosystems). The miRNA was purified and reverse transcribed using the TaqMan miRNA RT kit (Applied Biosystems) with miRNA-specific RT primers (Applied Biosystems). RT-PCR experiment was carried out on the StepOnePlus Real-time PCR platform (Applied Biosystems). The reaction mixture contained 0.2 ?M TaqMan probe, 2 ?l RT product, 10 ?M forward and reverse primers and 5 ?l TaqMan Universal PCR Master Mix. RNU6B was used as an endogenous control for hsa-mir-127-5p.  Cell transfection Cells in logarithmic phase were collected and trypsinized. The mature-type hsa-miR-127-5p (5?-CUGAAGCUCAGAGGGCUCUGAU-3?), hsa-miR-127-5p antisense inhibitor (anti–miR-127-5p) and nonspecific microRNA (miR-Control) were acquired from Dharmacon (Dharmacon, Lafayette, CO, USA). These oligonucleotides were transfected into glioma cells by using the Lipfactomine TM 2000 (Gibco BRL, Burlington, Ont., Canada) following the vendor-recommended transfection method. The transfection efficiency was assessed by performing a qRT-PCR experiment for the miRNA as described above.MTT assay The MTT assay was used to evaluate the viability of cells. The stock solution was prepared by dissolving 5 mg MTT reagent (Cell Proliferation Kit I, Roche, USA) in 1 mL PBS, followed by filtering in 0.22 ?m membrane. Cells were cultured for 48 h in 96-well plates.  Then, 20 ?L of stock solution (5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to each well and further incubated for an additional 4 h. After removal of supernatants, the plates were added with 150 ?L of DMSO (Sigma-Aldrich) for solubilization of the formazan crystals and the absorbance at 490 nm wavelength was measured using the microplate reader (Molecular Devices, Sunnyvale, CA, USA).Transwell migration and invasion assays To detect the extent of cell migration and invasion, transwell assay was performed. Matrigel-coated (invasion assay) or Matrigel-uncoated (migration assay) membranes were placed into the upper chambers at 37 °C overnight. Following lysis using trypsin, around 1×105 cells serum-free medium was employed for preparing dilutions. Next, cells were transferred into the upper chamber while the medium supplemented with 5 mg/L fibronectin and 10% FBS (Invitrogen) was added to the lower chamber. Following incubation in 5% CO2 incubator at 37 °C for 24 h, cells that passed through the membranes into the lower chamber were fixed for about 20 min using methyl alcohol and subsequently stained with crystal violet (0.1%) for 10 min. Finally, cells were examined using an optical microscope (Nikon, Japan) and counted. Luciferase reporter assay The mutagenesis kit was used to induce site-directed mutagenesis in REPIN1 3’UTR. Next, the wild-type and mutated REPIN1 3’UTR were inserted in the psiCHECK TM-2 vector (Promega, Madison, WI, USA) and the obtained recombinant psiCHECK TM-2 vectors were cotransfected into glioma cells with hsa-mir-127-mimics, hsa-mir-127 inhibitor or psiCHECK empty vector. Finally, the relative luciferase activity was determined after 48 h post-transfection with the Luciferase Assay Reagent II (Promega, Madison, WI, USA).Western blot Total protein was extracted using the radio-immunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St Louis, Mo) and separated using SDS–PAGE (Bio-Rad, Hercules, CA, USA) approach. Next, proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk for 1h. Then, the membrane was incubated with primary antibodies against REPIN1 (Thermo Fisher Scientific) and ?-actin (Thermo Fisher Scientific) at 4 °C for 12 h. Next, after incubation with anti-APS IgG-HRP (BOSTER) secondary antibodies, immune complexes were revealed by an enhanced chemiluminescent (ECL, Thermo Scientific). The relative protein expression was determined using a densitometric approach with Image J software. Statistical analysis Statistical analysis was achieved using the GraphPad Prism V6.01 software (GraphPad Software, Inc., La Jolla, CA, USA). The experiments were performed in triplicate and data expressed as average ± standard deviation (SD). The intergroup differences were evaluated using one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparison posttests. P < 0.05 was adopted for evaluation of statistical significance.RESULTS hsa-mir-127 is increased in glioma and is associated with poor survival   The online TCGA analysis indicated that hsa-mir-127 is expressed in different histological types of glioma, namely astrocytoma, oligoastrocytoma and oligo dendroglioma (Figure 1). Using RT-PCR to determine the expression of hsa-mir-127 in glioma cell lines U87 and LN-229, we found that hsa-mir-127 was markedly upregulated in these cells compared to normal brain glial cell HEB (Figure 2B). Bioinformatic analysis indicated that increased expression of hsa-mir-127 was associated with poor overall survival of patients (Figure 2C). Thus, hsa-mir-127, upregulated in glioma, is associated with poor survival of patients.Hsa-mir-127 induces the proliferation and inhibits the apoptosis of glioma cells In order to investigate the effects of hsa-mir-127 on the glioma cells, these cells were transfected with hsa-mir-127 mimics or hsa-mir-127 inhibitor or the negative control oligonucleotide (NC). The measurement of the transfection efficiency indicated that, compared to the NC group, transfection with hsa-mir-127 mimics group effectively increased hsa-mir-127 expression level (Figure 2A). In addition, hsa-mir-127 inhibitor significantly decreased the expression of hsa-mir-127 (Figure 2B), indicating the satisfactory transfection efficiency and its reliability for subsequent experiments.  MTT assay was performed to evaluate the effect of hsa-mir-127 on the proliferation of glioma cells. The results showed that relatively to the NC group, hsa-mir-127 mimics increased the proliferation of glioma cell lines U87 and LN-229 compared to control cels. On the contrary, transfection with the hsa-mir-127 inhibitor hindered the proliferation of both cell lines (Figure 2C). The flow cytometry analysis indicated that hsa-mir-127 mimics had a negative effect on the apoptosis of glioma cells while inverse effects were found with the inhibitor (Figure 2D). Thus, hsa-mir-127 induces the proliferation and inhibits the apoptosis of glioma cells. Hsa-mir-127 induces the migration and invasion of glioma cells Transwell migration assay indicated that transfection with hsa-mir-127 mimics induced the migration of U87 and LN-229 cells (Figure 3A) while inhibition of hsa-mir-127 inhibited the migration of these cells (Figure 3B). Similar results were found in transwell invasion assay. These results suggested that hsa-mir-127 promotes metastasis of glioma cells.Hsa-mir-127 targets REPIN1 and suppresses its expression in glioma cells     Examination of hsa-mir-127 association with mRNA expression in TCGA database indicated that hsa-mir-127 was positively and negatively associated with a multitude of genes (Figure 4A). Among genes that were the most negatively correlated with hsa-mir-127, we found REPIN1. A particular focus on REPIN1 clinical data in TCGA indicated that this gene is expressed in different histological types of glioma, including astrocytoma, oligoastrocytoma and oligodendroglioma while rare and decreased expression was recorded in glioblastoma multiforme histological type (Figure 4B). The Pearson correlation analysis indicated that REPIN1 and hsa-mir-127 were significantly and negatively correlated (Figure 4C). Furthermore, decreased expression of REPIN1 was associated with decreased overall survival of patients (Figure 4D). Using western blotting and RT-PCR, we found that, compared to normal glial cells, the expression of REPIN1 was downregulated in glioma cells at protein and mRNA levels (Figure 5A and 5B). In order to experimentally validate the correlation between hsa-mir-127 and REPIN1, the online Targetscan bioinformatics tool was first used to predict the target relationship between hsa-mir-127 and REPIN1. The result indicated that hsa-mir-127-5p has a putative binding site in the 3'-UTR of REPIN1 mRNA (Figure 5C). Luciferase reporter assay displayed that REPIN1 is a direct target of hsa-mir-127 (Figure 5D). In western blot analysis, the expression of REPIN1 was inhibited by hsa-mir-127 mimics but increased with the inhibitor (Figure 5E-5F). These results indicated that hsa-mir-127 directly targets REPIN1 and downregulates its expression in glioma.Hsa-mir-127 inhibits the tumor suppressor effects of REPIN1 in gliomas To investigate whether the hsa-mir-127/REPIN1 axis is involved in the development of glioma, REPIN1 was overexpressed in glioma cells overexpressing hsa-mir-127 and cells in NC group.  Comparatively to the NC group, hsa-mir-127 mimics induced the proliferation of glioma cells and REPIN1 overexpression partially reversed this effect (Figure 6A). Transwell assay equally displayed that hsa-mir-127 mimics induced the migration and invasion of gliomas cells but this effect was partially reversed by REPIN1 overexpression (Figure 6B-6C). These results suggested that hsa-mir-127 exerts its oncogenic effect in glioma, in part, by inhibiting REPIN1 expression.Discussion In this study, we investigated the regulatory interaction of hsa-mir-127 and REPIN1 in gliomas and the effects of this interaction on the proliferation, migration and invasion of glioma cells. Our results indicated that hsa-mir-127 is upregulated while REPIN1 expression is downregulated in gliomas. Both the upregulation of hsa-mir-127 and the downregulation of REPIN1 were associated with poor survival. Mechanistic studies indicated that REPIN1 is a direct target of Hsa-mir-127 and that the inhibitory effect of hsa-mir-127 on REPIN1 expression is partly involved in the development and metastasis of gliomas. Though hsa-mir-127 dysregulation has been conveyed in different cancer types, its expression and mechanism are controversial. In most cases, hsa-mir-127 is reported as a tumor suppressor. It was previously reported that mir-127 exerts anti-tumor effects in epithelial ovarian cancer via modulation of BAG5 expression (Bi et al., 2016; Yu et al., 2016). Overexpression of miR-127-3p was also found to inhibit the tumor development by regulating COA1, GLE1 and PDIA6 in the giant cell tumor of bone derived stromal cells (Fellenberg et al., 2016; Herr et al., 2017). In the esophageal squamous cell carcinoma, mir-127 was found as a tumor suppressor due to its negative regulation of the oncogene FMNL3 (Gao et al., 2016). The tumor suppressor roles of mir-127-5p was equally found in hepatocellular carcinoma cells essentially through its targeting of the biliverdin reductase B/nuclear factor-kappaB pathway (Huan et al., 2016). In other studies, mir-127 was found to induces Adriamycin resistance by regulating cell cycle arrest and apoptosis in glioma cells (Feng & Dong, 2015). Previous studies equally indicated that mir-127-3p promotes the metastatic phenotypes of glioblastoma cells and hepatocellular carcinoma cells through targeting SEPT7 (Jiang et al., 2014; Zhou et al., 2014). However, the role of hsa-mir-127 in glioma has not been fully investigated and further elucidation experiments are required. In the present study, in addition to the increased expression of hsa-mir-127 in glioma, we observed that hsa-mir-127-5p promoted the metastatic features of gliomas, which displayed that hsa-mir-127-5p could be a biomarker and therapeutic target for gliomas. This assumption requires a scrupulous understanding of hsa-mir-127-5p related mechanisms. REPIN1, a 60 kDa origin-specific DNA-binding protein, is known to facilitate DNA bending in the vicinity of gene origin of replication (Caddle et al., 1990). Functional studies indicated that REPIN1 promotes the migration and epithelial-mesenchymal transition (EMT) of breast cancer cells by regulating p53 (Chen & Chiu, 2015). In colorectal carcinoma, REPIN1 is associated with distant metastasis and poor survival by directly suppressing the expression of miR-15a/16-1(Shi et al., 2014). Moreover, in non-small cell lung cancer, findings suggest that REPIN1 may inhibit the TGF-?1-induced SMAD2 phosphorylation to subsequently induce cell growth in NSCLC (Wang et al., 2015). The role of REPIN1 in gliomas has not been conveyed so far. In this study, we focused on finding and elucidating the correlation between REPIN1 and hsa-mir-127 and the function of this axis in glioma development. The results demonstrated that hsa-mir-127 promotes its oncogenic effects in glioma by directly targeting REPIN1. Previous studies demonstrated that REPIN1 regulates DNA synthesis in mammalian cells (Dailey et al., 1990) and may act as an accessory component in the recognition of the origin of replication before the assemblage of pre-initiation complexes (Houchens et al., 2000). Thus, we hypothesized that the effect of REPIN1 on the metastatic phenotype of glioma cells may occur through modulation of replication related genes and cell cycle. Nevertheless, our study presents some limitations. Firstly, the mechanisms underlying the regulatory network of REPIN1 needs to be further elucidated in future studies to better elucidate the molecular networking of and functional role of REPIN1 in glioma cells. Moreover, since TCGA showed the association of hsa-mir-127 with a multitude of gene, further studies are critical in better understanding its role in glioma physiopathology.  In conclusion, we demonstrated the oncogene role of hsa-mir-127 and that of its inhibitory effect on its target gene REPIN1 in glioma. Especially, our work revealed that hsa-mir-127 induced the proliferation migration and invasion of glioma cells by, partly, repressing the tumor suppressor role of REPIN1 in glioma.