Increased MiR-130a In Glioma Directly Targets SMAD4and Is Involved In Modulating Cell Proliferation And Apoptosis

Gliomas are the most common and the most malignant brain tumors, accouting for45~55%of all intracranial tumors. The incidence of glioma worldwide is about6~12per100,000. According to the criteria from the World Health Organization (WHO), humangliomas are divided into four grades on the basis of their histology and prognosis, withhighest grade glioma being represented by grade IV (glioblastoma multiforme, GBM).GBM is also the most lethal type of human brain cancer in adults, with at least40,000newcases per year worldwide and a median survival less than one year. Current therapies formalignant glioma, though they are very aggressive and usually involve a combination ofsurgery, radiotherapy and chemotherapy, have not been successful, due to several factors.These include rapidness and invasiveness of tumor growth, the drug resistance tochemotherapy and the genetic heterogeneity of the tumors.The occurrence anddevelopment of glioma are thought to be multifactorial. The activation of the oncogenesand the inactivation and/or loss of the tumor suppressor genes are the most primarymolecule event. The clinical significance of molecular parameters for the diagnostic and prognostic assessment of gliomas is still limited. Therefore further investigation of themolecular mechanisms underlying oncogenesis and progression of these most commonbrain tumours is necessary for researchers to improve the diagnostic assessment and todevise novel, individually tailored therapeutic approaches.MicroRNAs (miRNAs) are a class of endogenously expressed small noncodingRNAs which are usually21-23nucleotides long. miRNAs regulate gene expression andplay important roles in a variety of physiological and pathological processes, such as cellproliferation, differentiation and apoptosis. To date,1921human miRNAs have beenidentified (miRBase Release18, November2011). Growing evidence has shown thatmiRNAs are frequently dysregulated in human cancers and can act as both tumorsuppressors and oncogenes. Along with the discovery of microRNA, more and moreresearch focusing on its relationship with glioma was carried out to investigate thebiological features of glioma and to provide experimental evidence for novel therapeutics.Ciafréet al. examined by microarray the global expression levels of245microRNAsin glioblastoma multiforme, and found miR-130a was up-regulated in5out of9samples(2.11-to5.3-fold change). Here we investigated the expression of miR-130a in37gliomaspecimens,6normal brain tissues and2cell lines by quantitative real-time PCR. Theresults revealed that miR-130a was significantly up-regulated in about78%of gliomatissues and2glioma cell lines examined, compared with normal brain tissues. Then wetransfected miR-130a inhibitors into human glioma U251cells and U87cells by usingLipofectamineT M2000to observe its effects on cell proliferation and apotosis. On thepremise of good transfection efficiency, cell proliferation was analyzed using dimethylthiazolyl diphenyltetrazolium (MTT) assay and cell apoptosis were studied by flowcytometry (FCM). The results showed that miR-130a inhibitors inhibited U87cells in atime-dependent manner, with growth-inhibitory effects being apparent as early as thesecond day. Likewise, U251cells growth was also inhibited in a time-dependent manner,with growth inhibition being observed from the third day.The apoptotic rates of both U251and U87cells transfected with miR-130a inhibitors were significantly higher than cellstransfected with scrambled control and cells untransfected. SMAD4(mothers against decapentaplegic homologue4), located on chro mosome18q21.1, was initially isolated as a tumor suppressor gene in pancreatic ductaladenocarcinomas. SMAD4, also termed DPC4(deleted in pancreatic cancer locus4), was a2680-bp transcript and contained11exons and10introns. SMAD4is now considered as anovel tumor suppressor gene and has influence on the development and progression ofvarious human tumors. Being a major transducer of TGF-β, BMP and Activin signaling,SMAD4may regulate cell proliferation, differentiation and extracellular matrix prod uction.Professor He Shiming of our department has previously investigated the expression ofSMAD4in252cases of human glioma and compared the expression with the patients′survival rates. The data demonstrated that both SMAD4mRNA and protein was decreasedin glioma compared to normal brain tissue, and the reduced expression of SMAD4wascorrelated with poor outcome, indicating that SMAD4may play an inhibitive role duringthe development of glioma and may be a potential prognosis predictor of glioma. Acomputational analysis with the use of TargetScan predicted that SMAD4mRNA containstwo miR-130a-binding sites.To confirm the regulation of miR-130a to SMAD4in glioma, we performedluciferase assay, quantitative real-time PCR and western blotting with the use of theconstructed pMIR-REPORT vectors which were kindly provided by Mattias H ger fromDenmark. A pronounced reduction of luciferase activity was observed in cells transfectedwith plasmid containing both miR-130a binding sites, whereas plasmid containing onepredicted site and one mutated site showed less reduction of luciferase activity. No effectwas seen from the construct with both sites mutated.The SMAD4mRNA expression levelof cells transfected with miR-130a mimics showed no significant difference with that ofcontrol cells. Western blotting assays indicated that the SMAD4protein level wasmarkedly reduced in cells overexpressing miR-130a compared with the scrambled control,whereas the SMAD4protein level was increased in cells transfected with miR-130ainhibitors.Together these results indicated that miR-130a can bind to the2predicted targetsites inSMAD43′UTR and regulate SMAD4by repressingpost-transcriptional translationrather than by mediating mRNA cleavage. In conclusion, our research confirmed miR-130a expression was increased in humanglioma. Transfection of its inhibitors into glioma cells could inhibit the cells proliferationand promote cells apoptosis.This preliminary research identified miR-130a as one of theoncogenes in human glioma, implicating that it may be a candidate target for gliomatherapy. Furthermore, we found that miR-130a could negatively regulate the expression ofSMAD4gene in glioma cells, providing the evidence for further explore about themolecular pathologic mechanism and potential therapeutic strategy for human glioma.