MiR-130a Inhibits Cell Growth And Epithelial To Mesenchymal Transition By Repression Of HMGB2 In Glioma

Background and ObjectivesGlioma is the most comon primary ntracranial tumor.the outcome were still poor using all the current therapeutic.Hence,identification and characterization of the regulatory molecules that involved in the glioma tumorigenesis may offer important targets for treatment strategies.MicroRNAs(miRNAs),a class of endogenous non-coding RNAs that are 19-25 nucleotides in length,Previous study reveals that miR-130a could serve as prognostic and predictive markers for survival of glioma patients.However,the in vivo function and underlying mechanism of miR-130a in glioma still have not been clarified clearly.High-mobility group box-2(HMGB-2)belongs to the HMG protein families,In glioma,HMGB2 is associated with prognosis of glioblastoma by promoting cell viability,invasion,and chemotherapeutic resistance.In this study,the expression of miR,130a in gliomas was detected,and lentivirus miR-130a was stably overexpressed in glioma cell lines to detect the changes of cell proliferation,invasion and EMT phenotype.miR-130a-mediated molecular mechanism is a basis to find a new target for glioma treatment.Contents and methods1.Identification the expression of miR-130a in gliomaThe mRNA levels of miR-130a were measured respectively by RT-PCR in glioma cells and tissues and normal brain tissues.2.Functional analysis of miR-130a in glioma in vitrolentivirus miR-130a was transfected into U87 glioma cell lines in vitro,with si-Scrambled treated as a negative control group(NC).The proliferation,invasion,cell cycle were examined separately by MTT,Boyden chambers assay,and FACS caliber flow cytometry in vitro.Subcutaneous tumor formation in nude mice was used to verify that miR-130a inhibits the proliferation of glioma cells.3.Priliminary investigation of molecular basis mediated by miR-130a in gliomaAs the expression of miR-130a was over-expressed,the invasion ability of cells was inhibited.Therefore,we detected the expression of E-cadherin,N-cadherin,vimentin and fibroin in cell invasion by immunofluorescence.E-cadherin,N-cadherin,Vimentin protein expression.Meanwhile,Western blot was used to quantitatively detect the expressions of CDK4,c-myc,and Cyclin D1 in G1/S phase after miR-130a overexpression.Bioinformatics software predicts that HMGB2 may be a direct target gene of miR-130a.The expression of HMGB2 inhibited by miR-130a was dected by fluorescence quantitative PCR and Western blot.Dual luciferase gene reporter assay was used to demstreate miR-130a can be targeted to bind HMGB2 mRNA in the 3’UTR region and thus inhibit the expression of HMGB2 gene;MTT experiment and clone formation experiments confirmed that miR-130a inhibit the glioma cell proliferation by targeting HMGB2;Boyden chamber invasion assay was used to dectected the miR-130a inhibit the invasion of glioma cells by targeting HMGB2.4.The molecular mechanism of EZH2 inhibiting miR-130a transcriptionWe used a combination of bisulfite restriction endonuclease assay to dectet DNA methylation levels;informatics analysis showed that EZH2 can be combined with the promoter region of miR-130a;using chromatin immunoprecipitation in vivo conditions further verification EZH2 and miR-130a promoter region;EZH2 was silenced in U87 glioma cells by RNA interference and then the miR-130a expression was detected by RT-PCR;We further treated the U87 cells with deazaneplanocin A(DZNep)and then detect the miR-130a expression level.ResultsWe demonstrated that miR-130a expression was frequently downregulated in glioma specimens and cell lines.Functionally,re-expression of miR-130a effectively reduced glioma cell proliferation and invasion.Mechanistically,we confirmed that the signal transducer and activator of transcription 3(HMGB2)3’-UTR was a putative target of miR-130a,and that re-expression of HMGB2 abrogated miR-130a function in glioma cells.Furthermore,we found that HMGB2 expression negatively correlated with that of miR-130a in human glioma tissues.Subsequently,we asked whether miR-130a down-regulation in glioma cells was due to promoter methylation.Bisulfite sequencing PCR(BSP)revealed that miR-130a methylation was higher in glioma cell lines compared with NBC.We observed that H3K27me3 was steadily enriched at the miR-130a promoter.EZH2 down-regulation or treatment with deazaneplanocin A(DZNep)increased miR-130a levels,supporting the notion that miR-130a was epigenetically silenced by DNA methylation and EZH2-mediated histone methylation in glioma.ConclusionOverall,our results provide the first evidence that the EZH2/miR-130a/HMGB2 axis controls growth and invasion in glioma cells.Because down-regulation of miR-130a is associated with a poor prognosis and the restoration of miR-130a decreased cell growth and invasion,targeting miR-130a expression might be a potential glioma treatment.