ABSTRACT efficacy of the therapy effect against glioma. In

ABSTRACT This work aimed toscrutinize the inter-regulatory functions of hsa-mir-127 and replicationinitiator 1 (REPIN1) on the proliferation and metastasis of glioma cells. The in-silico data on the implication hsa-mir-127and REPIN1 in glioma were retrieved from The Cancer Genome Atlas (TCGA). Theexpression levels of hsa-mir-127 and REPIN1 mRNA were determined by qRT-PCRwhereas western blot was used detection of REPIN1 protein expression in gliomacell lines.

The proliferation of glioma cells was determined by means of the MTTassay, while the transwell assay was employed for assessing the extent of cellmigration and invasion. The interaction among REPIN1 and hsa-mir-127 was checkedusing the luciferase reporter assay. The expression of hsa-mir-127 was markedlyincreased in clinical data obtained from TCGA and in glioma cells compared withnormal tissues and control cells, respectively.

Increased expression ofhsa-mir-127 and decreased expression of REPIN1 were both associated with pooroverall survival. Moreover, hsa-mir-127 overexpression noticeably promoted the proliferation,inhibited apoptosis and increased the invasive and migratory capacities ofglioma cells. Inverse effects were found with hsa-mir-127 antisense inhibitor. Interestingly,overexpressing hsa-mir-127 downregulated REPIN1 expression and luciferasereporter assay displayed that the tumorigenesis effect of hsa-mir-127 requires,in part, its direct targeting of REPIN1.

Conclusively, the hsa-mir-127/REPIN1 pathwayis involved in gliomas and could be a potential therapeutic target.   Keywords: glioma, hsa-mir-127,REPIN1, proliferation, invasion, migration INTRODUCTION Gliomas are themost common brain tumors in children and adolescents (Ostrom et al., 2015).Low-grade (benign) gliomas are more common in younger age groups, whilemalignant gliomas affect older children or adolescents (de Groot, 2015).These tumors include very good prognosis forms such as pilocytic astrocytomawith types much more difficult to treat such as infiltrating glioma of thebrainstem.

Surgery is usually used as a first-line treatment for malignantastrocytomas whereas most of the treatment protocols for malignant gliomainclude radiotherapy, chemotherapy, and new forms of treatment, such asphotoradiotherapy with porphyrins that are being assessed (Agulnik & Mason, 2006).Nonetheless, the aggressive property of malignant gliomas thwarts the efficacyof the therapy effect against glioma. In addition, the molecular mechanisms involvedin gliomas physiopathology are not fully elucidated, which is a key drawback tothe development of efficient anti-glioma drugs. Thus, scrutinizing the molecularmechanisms governing the proliferation, migration and invasion of glioma cellsis of paramount significance.

Micro RNAs (miRNAs)are approximately 21 nucleotides-length noncoding small RNAs that control theregulation of their target genes. Increasing number of studies reveals the correlationof miRNAs with human disorders including cancers. Hsa-mir-127 is a miRNA withcontroversial roles in different type of human cancers. Indeed, some studies onovarian, gastric, esophageal, giant bone, pancreatic and hepatocellular cancershave demonstrated that hsa-mir-127 exhibits a tumor suppressor function (Bi et al., 2016; Fellenberg et al., 2016; Gao et al.

,2016; Guo et al., 2013; Herr et al., 2017; Huan et al., 2016; Yu et al., 2016;Zhang et al., 2016; Zhou et al., 2014)whereas other studies indicated the oncogene role of this miRNA inglioblastoma, lung, breast, and mucoepidermoid  carcinoma cells (Jiang et al.

, 2014; Shi et al., 2017; Shin et al.,2013; Wang et al., 2014; Yan et al., 2008).

Though silencing of has-miR-127 in glioma cells was conveyed to counteractAdriamycin resistance through mechanisms involving cell cycle arrest andinduction of cell apoptosis (Feng & Dong, 2015),the functional role of has-miR-127 has not been fully investigated and thepotential molecular mechanisms need to be elucidated.Replicationinitiator 1 (REPIN1) is a zinc finger DNA-binding protein that enables theinitiation of DNA replication. The physiological and regulatory roles of REPIN1have not been extensively studied since its discovery but recent findings have suggestedits implications in obesity and related metabolic syndromes including glucosetransport, fatty acid transport, adipogenesis andthe formation and fusion of lipid droplets (Andrade Fde et al., 2015; Bahr et al., 2011a; Bahr etal.

, 2011b; Heiker & Kloting, 2013; Hesselbarth et al., 2017; Kern et al.,2014; Kloting et al., 2007; Kunath et al., 2016; Ruschke et al., 2010).

Given that REPIN1 is involved in DNA replication, its dysregulation may be apotential mechanism involved in tumorigenesis. Up to now, only one study hassuggested the association of REPIN 1 in breast cancer (Rengasamy et al., 2017).However, the functional role of REPIN1 and its probable regulation by miRNAs ingliomas are still unknown and need to be deeply scrutinized.

Hence, in thisstudy, we aimed to explore the implication of has-miR-127 and REPIN1 in gliomaprocesses and the possible regulatory interaction among them. In silico studiesindicated that hsa-mir-127 high expression was associated with decreasedsurvival of glioma patients while the inverse was observed with REPIN1. Theeffect of hsa-mir-127 on the proliferation, invasion and migration of gliomacells was investigated and REPIN1 was proposed as the target gene of hsa-mir-127.MATERIALS AND METHODS Retrieval of has-mir-127 and REPIN1 from TCGA glioma dataThe freelyavailable portal LinkedOmics (www.linkedomics.org) that containsmulti-omics data from the 32 TCGA Cancer types was adopted for uncovering the effectof has-mir-127 and REPIN1 on the survival of gliomapatients and their corresponding expression profiles in different tissue types.The LinkFinder analytical module was used to search for mRNA expressionsignatures (from mRNAseq data) associated with has-mir-127. The Pearsoncorrelation analysis was applied to assess the correlation of has-mir-127 with mRNAexpression signatures.

Analysis results were visualized by scatter plots, boxplots, or Kaplan-Meier plots.    Cell lines and culture The normal humanglial cell HEB and two glioma cell lines U87 and LN-229 were al purchased from ChineseAcademy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified eaglemedium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, SouthLogan, UT, USA), 50 ug/mL streptomycin and 100 ug/mL penicillin at 37 °C in anincubator containing 5% CO2. Quantitative real-time PCR Total RNA was isolatedwith TRIzol reagent (Invitrogen, Carlsbad, CA, USA). An aliquot of purified RNAwas reverse-transcribed into cDNA using the Reverse Transcription Kit (TakaraBio, Inc.

, Otsu, Japan) in accordance with the manufacturer’s instructions. Theamplification of REPIN1 was performed on a Biorad Realtime PCR platform. The PCRreaction cycling conditions for the 30 cycles were: 94 °C for 2 min, 94 °C for30 s, 56 °C for 30 s, 72 °C for 1 min and 72 ? for 10 min. GAPDH was used as anendogenous 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 expressionlevel of mature hsa-mir-127-5p was determined with a TaqMan miRNA assay kit(Applied Biosystems). The miRNA was purified and reverse transcribed using theTaqMan miRNA RT kit (Applied Biosystems) with miRNA-specific RT primers(Applied Biosystems). RT-PCR experiment was carried out on the StepOnePlusReal-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 ?lTaqMan Universal PCR Master Mix. RNU6B was used as an endogenous control for hsa-mir-127-5p. Cell transfection Cells inlogarithmic phase were collected and trypsinized. The mature-type of hsa-miR-127-5p,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 LipfactomineTM 2000 (Gibco BRL, Burlington, Ont., Canada) following the vendor-recommended transfectionmethod. The transfection efficiency was assessed by performing a qRT-PCRexperiment for the miRNA as described above.

MTT assay The MTT assay was usedto evaluate the viability of cells. The stock solution was prepared bydissolving 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 in96-well plates.  Then, 20 ?L of stocksolution (5 mg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to each well andfurther incubated for an additional 4 h. After removal of supernatants, the plateswere added with 150 ?L of DMSO (Sigma-Aldrich) for solubilization of theformazan crystals and the absorbance at 490 nm wavelength was measured usingthe microplate reader (Molecular Devices, Sunnyvale, CA, USA).Transwell migration and invasion assays To detect theextent of cell migration and invasion, transwell assay was performed.

Matrigel-coated(invasion assay) or Matrigel-uncoated (migration assay) membranes were placedinto the upper chambers at 37 °C overnight. Following lysis using trypsin, around1x105 cells serum-free medium was employed for preparing dilutions.Next, cells were transferred into the upper chamber while the mediumsupplemented with 5 mg/L fibronectin and 10% FBS (Invitrogen) was added to the lowerchamber. Following incubation in 5% CO2 incubator at 37°C for 24 h, cells that passed through the membranes into the lower chamberwere fixed for about 20 min using methyl alcohol and subsequently stained withcrystal violet (0.

1%) for 10 min. Finally, cells were examined using an opticalmicroscope (Nikon, Japan) and counted. Luciferase reporter assay The PhusionSite-Directed Mutagenesis Kit (Thermo Fisher Scientific) was used to induce mutagenesisin REPIN1 3’UTR. Next, the wild-type and mutated REPIN1 3’UTR were inserted inthe psiCHECK TM-2 vector (Promega, Madison, WI, USA) and the obtainedrecombinant psiCHECK TM-2 vectors were cotransfected into glioma cells with hsa-mir-127-mimics,hsa-mir-127 inhibitor or psiCHECK empty vector. Finally, the relativeluciferase activity was determined after 48 h post-transfection with theLuciferase Assay Reagent II (Promega, Madison, WI, USA).Western blot Total protein was extractedusing 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 wasincubated with primary antibodies against REPIN1 (Thermo Fisher Scientific) and?-actin (Thermo Fisher Scientific) at 4 °C overnight. Next, after incubationwith anti-APS IgG-HRP (BOSTER) secondary antibodies, immune complexes were revealedby an enhanced chemiluminescent (ECL, Thermo Scientific). The relative proteinexpression was determined using a densitometric approach with Image J software.Statistical analysis Statisticalanalysis was achieved using the GraphPad Prism V6.01 software (GraphPadSoftware, Inc., La Jolla, CA, USA). The experiments were performed intriplicate and data expressed as average ± standard deviation (SD).

Theintergroup differences were evaluated using one-way ANOVA or two-way ANOVAfollowed by Tukey’s multiple comparison posttests. P < 0.05 was adopted forevaluation of statistical significance.

RESULTS hsa-mir-127 is increasedin glioma and is associated with poor survival The online TCGA analysisindicated that hsa-mir-127 is expressed in different histological types ofglioma, namely astrocytoma, oligoastrocytoma and oligo dendroglioma (Figure 1).Using RT-PCR to determine the expression of hsa-mir-127 in glioma cell lines U87and LN-229, we found that hsa-mir-127 was markedly upregulated in these cellscompared to normal brain glial cell HEB (Figure 2B).Bioinformatic analysis indicated that increased expression of hsa-mir-127 wasassociated with poor survival of patients (Figure 2C). Consequently, hsa-mir-127, upregulated in glioma, is associatedwith poor survival of patients.

Hsa-mir-127 induces the proliferation of glioma cells In order toinvestigate the effects of hsa-mir-127 on the glioma cells, these cells weretransfected with hsa-mir-127 mimics or hsa-mir-127 inhibitor or the negativecontrol oligonucleotide (NC). The measurement of the transfection efficiency indicatedthat, compared to the NC group, transfection with hsa-mir-127 mimics group effectivelyincreased hsa-mir-127 expression level (Figure 2A). In addition, hsa-mir-127inhibitor significantly decreased the expression of hsa-mir-127 (Figure 2B),indicating the satisfactory transfection efficiency and its reliability forsubsequent experiments.  MTT assay wasperformed to evaluate the effect of hsa-mir-127 on the proliferation of gliomacells. The results showed that comparatively to the NC group, hsa-mir-127 mimics increased the proliferation of gliomacell lines U87 and LN-229 compared to control cells. On the contrary, transfectionwith the hsa-mir-127 inhibitor hindered the proliferation of both cell lines(Figure 2C).

As a result, hsa-mir-127 triggers the proliferation of gliomacells. Hsa-mir-127 induces the migration and invasion ofglioma cells Cell migration assayindicated that transfection with hsa-mir-127 mimics promoted the migration of U87and LN-229 cells (Figure 3A) while inhibition of hsa-mir-127 inhibited themigration of these cells (Figure 3B). Similar results were found in cell invasionassay. These results suggested that hsa-mir-127 promotes the metastaticphenotype of glioma cells.REPIN1 is a direct target of its negative regulator hsa-mir-127in glioma cells Examination ofhsa-mir-127 association with mRNA expression in TCGA database indicated that hsa-mir-127 was positively and negatively associatedwith a multitude of genes (Figure 4A). Among genes which were the mostnegatively correlated with hsa-mir-127, we found REPIN1. A particular focus on clinicaldata in TCGA indicated that REPIN1 is expressed in different histological typesof glioma, including astrocytoma, oligoastrocytoma and oligodendroglioma whilerare and decreased expression was recorded in glioblastoma multiformehistological type (Figure 4B).

The Pearson correlation analysis indicated thatREPIN1 and hsa-mir-127 were significantly and negatively correlated (Figure4C). Furthermore, decreased expression of REPIN1 was associated with decreasedoverall survival of patients (Figure 4D). Using westernblotting and RT-PCR, we found that, compared to normal glial cells, theexpression of REPIN1 was downregulated in glioma cells at both mRNA and proteinlevels (Figure 5A and 5B). In order to experimentally validate the regulatory correlationbetween hsa-mir-127 and REPIN1, the online Targetscan tool was first used to predictthe target binding relationship amid hsa-mir-127 and REPIN1.

The result indicated that hsa-mir-127-5p has a putative binding site in the 3′-UTRof REPIN1 (Figure 5C). Luciferase reporter assay displayed that 3′-UTR ofREPIN1 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 withthe inhibitor (Figure 5E-5F). These results indicated that hsa-mir-127 directlytargets REPIN1 and downregulates its expression in glioma.

Hsa-mir-127 inhibits the tumor suppressor effects of REPIN1in gliomas To investigatewhether the hsa-mir-127/REPIN1 axis is involved in the development of glioma,REPIN1 was overexpressed in glioma cells transfected with hsa-mir-127 mimics andcells in NC group.  Relatively to the NCgroup, hsa-mir-127 induced the proliferation of glioma cells whereas REPIN1 partlyabrogated this effect (Figure 6A). Transwell assay equally displayed that hsa-mir-127mimics induced the migration and invasion of glioma cells but this action was partlybacked up by REPIN1 overexpression (Figure 6B-6C). These results suggested thathsa-mir-127 exerts its oncogenic effect in glioma, in part, by inhibiting REPIN1.Discussion In this study, we investigatedthe regulatory function of hsa-mir-127 and REPIN1 in gliomas and the effects ofthis interaction on the proliferation, migration and invasion of glioma cells. Ourresults indicated that hsa-mir-127 is upregulated while REPIN1 expression isdownregulated in gliomas. Both the increased expression of hsa-mir-127 and the decreasedexpression of REPIN1 were associated with poor survival. Mechanistic studiesindicated that REPIN1 is a direct target of hsa-mir-127 and that inhibition ofREPIN1 expression by hsa-mir-127 is partly involved in the development andmetastasis of gliomas.

Though theimplication of hsa-mir-127 has been conveyed in different cancer types, itsexpression and mechanism are controversial. In most cases, hsa-mir-127 isreported as a tumor suppressor. Indeed, previously reported findings indicate thatmir-127 exerts anti-tumor effects in epithelial ovarian cancer via modulating BAG5expression (Bi et al., 2016; Yu et al., 2016).Overexpression of miR-127-3p was also found as tumor suppressor by controlling COA1,GLE1 and PDIA6 in the giant cell tumor of bone derived stromal cells (Fellenberg et al., 2016; Herr et al.

, 2017).In esophageal squamous cell carcinoma, mir-127 was found to exert tumorsuppressor activity via its negative regulation of FMNL3 (Gao et al., 2016).The tumor suppressor roles of mir-127-5p was equally found in hepatocellularcarcinoma cells essentially through its targeting of the biliverdin reductaseB/NF-?B pathway (Huan et al., 2016).In other findings, mir-127 was found to promote Adriamycin resistance in gliomacells through regulating cell apoptosis (Feng & Dong, 2015).Previous studies equally disclosed that mir-127-3p improves the proliferation, migrationand invasion of glioblastoma cells and hepatocellular carcinoma cells through targetingSEPT7 (Jiang et al.

, 2014; Zhou et al., 2014).However, the function of hsa-mir-127 in the pathophysiology of glioma has notbeen fully investigated and further elucidation experiments are required.

Inthe present study, in addition to the increased expression of hsa-mir-127, we noticedthat hsa-mir-127-5p increased the invasive ability of glioma cells, which impliedthat hsa-mir-127-5p could be a therapeutic target for gliomas. This statementrequires a scrupulous understanding of hsa-mir-127-5p related mechanisms. REPIN1, a 60 kDaorigin-specific DNA-binding protein, is known to facilitate DNA bending in thevicinity of gene origin of replication (Caddle et al., 1990).Functional studies indicated that REPIN1 promotes the migration and epithelial-mesenchymaltransition (EMT) of breast cancer cells by regulating p53 (Chen & Chiu, 2015).In colorectal carcinoma, REPIN1 is associated with distant metastasis and poorsurvival 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 mayinhibit the TGF-?1-induced SMAD2 phosphorylation to subsequently induce cellgrowth in NSCLC (Wang et al.

, 2015).The role of REPIN1 in gliomas has not been conveyed so far. In this study, we focusedon finding and elucidating the correlation between REPIN1and hsa-mir-127 and the function of this axis in glioma development. Theresults demonstrated that hsa-mir-127 promotes its oncogenic effects in gliomaby directly targeting REPIN1. Previous studies demonstrated that REPIN1 regulatesDNA synthesis in mammalian cells (Dailey et al., 1990)and may act as an accessory component in the recognition of the origin ofreplication before the assemblage of pre-initiation complexes (Houchens et al., 2000).

Thus, we hypothesized that the effect of REPIN1 on themetastatic phenotype of glioma cells may occur through modulation ofreplication related genes and cell cycle. Nevertheless, ourstudy presents some limitations. Firstly, the mechanisms behind the REPIN1 regulationpathways needs to be further elucidated in future works to better elucidate themolecular 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 demonstratedthe oncogene role of hsa-mir-127 and that of its inhibitory effect on itstarget gene REPIN1 in glioma.

Especially, our work revealed that hsa-mir-127induced the proliferation migration and invasion of glioma cells by, partly, repressingthe tumor suppressor role of REPIN1 in glioma.