An Experimental Study On The Roles Of MiRNA-126 And MiRNA-34a In Glioma Cells

Gliomas account for approximately 50% of all central nervous system tumors, and these leisions are locally invasive and recur even after aggressive resection, radiation, and chemotherapy are performed.The prognosis for patients with high-grade gliomas,which include anaplastic astrocytoma(WHOâ…¢) and glioblastoma multiforme(GBM,WHOâ…£)), remains dismal.Hence,there is a great hope for novel therapeutic approaches.MicroRNA(miRNA) are a family of approximately 22-nuleotide-long,highly conserved, non-coding, single-stranded small RNAs, which induce target gene silence by inhibiting translation or by targeting messenger RNA (mRNA) for degradation in a post-transcriptional fashion.MiRNAs involved in a series of biological events, including development, hematopoiesis,organ generation,apoptosis,development and progress of cancers. Currently, accumulating evidence has suggested that aberrant expressions of microRNAs are strongly implicated in tumorigenesis of gliomas. MiRNAs, involved in the pathogenesis of gliomas, can act as tumor suppressors when down-regulated, as are the cases of miR-128, miR-124,miR-137,miR-7,miR-125b, miR-15b or as oncogenes when over-expressed,as are the cases of miR-21 and miR-221-222.This study is to observe the antitumor effects of miR-126 and miR-34a on human glioma cell lines.The results would establish the groundwork for the future clinical studies and application.1. Based on previouly-reported miRNA expression proflile of glioma cell lines, miR-126 was chosen as the subject.Expression levels of miR-126 in normal brains, glioma tissues of WHOâ…¡,â…¢,â…£,and glioma cell lines were detected using qRT-PCR.Human U251,A172, SHG-44 glioma cells were transfected with miR-126 mimics,and the effects of miR-126 over-expression were assessed by MTT assays,cell cycle analysis,caspase-3 activation,and in vitro migartion and invasion assays. A computational search revealed a conserved target site of miR-126 within the 3'-untranslated region of Crk. Luciferase reporter assay was performed to examine the effects of miR-126 on expression of potential target gene Crk,and mRNA and protein expression of Crk after miR-126 (?) analysis.2. Expression levels of miR-34a in normal brains, p53 mutant or p53 wild-type glioma cell lines were detected using qRT-PCR.P53 mutant human U251 glioma cells were transfected with miR-34a mimics.and the effects of miR-34a restoration were assessed by MTT assays.cell cycle analysis,caspase-3 activation.and in vitro migartion and invasion assays. A bioinformatic search revealed SIRT1 was a potential downstream target,which was controversial in the regulating way of miR-34a in previous documentation. Luciferase reporter assay was performed to examine the effects of miR-34a on expression of SIRT1,and mRNA and protein expressions of SIRT1 after miR-34a transfection were detected by qRT-PCR and western blotting analysis.3.Bcl-2 protein expressions were detected by WB after transfection of miR-34a mimics or Bcl-2 siRNA;IC 50 data were calculated by MTT in combination of miR-34a or Bcl-2 siRNA and chemotherapeutic agents (ACNU;VM-26;VCR; CDDP).And then,the protein expressions of Bcl-2 in blank group,miR-34a group, chemotherapeutic agents group,miR-34a+chemotherapeutic agents group were assayed by WB.1. Quantitive real-time RT-PCR analysis revealed that miR-126 expression was markedly reduced in all-grade glioma tissues and glioma cell lines U251,A172 and SHG44, versus normal brain tissues (p<0.05). Ectopic expression of miR-126 by transfection of miR-126 mimics had no effects on cell growth, cell cycle distribution or apoptosis, but significantly decreased in vitro migration and invasion capabilities of glioma cells (A172, p<0.01;U251 and SHG-44, p<0.05). Furthermore, luciferase reporter assays revealed that Crk, involved in cell growth, motility, differentiation, and adhesion, was a functional target of miR-126. Also, over-expression of miR-126 decreased Crk protein levels but not mRNA, which demonstrated miR-126-induced Crk inhibition occurred at the post-transcriptional level.2. miR-34a expression was markedly reduced in p53-mutant cells U251 compared with A172 and SHG-44 cells expressing wild-type p53 and normal brains (p<0.01) Over-expression of miR-34a in U251 cells resulted to inhibition of cell growth and arrest in G0-G1 phase, and induced apoptosis.Also,restoration of miR-34a significantly reduced in vitro migration and invasion capabilities (p<0.01).Reporter assays indicated that SIRT1 was a direct target of miR-34a.In U251 cells, over-expression of miR-34a decreased SIRT1 protein levels but not mRNA expressions, which demonstrated miR-34a-induced SIRT1 inhibition occurred at the post-transcriptional level.3.Both of miR-34a and Bcl-2 siRNA inhibited the expression of Bcl-2 protein.IC50 of combination of miR-34a and chemotherapeutics(ACNU; VM-26;VCR;CDDP) in U251 glioma cells were 103.1,3.4,0.8,4.3 ug/ml;while IC50 of combination of NC and chemotherapeutics were 305.4,5.7,1.8,7.8 ug/ml. miR-34 restoration in U251 cells rendered the cells 2-3-fold more sensitive to the four chemotherapeutic agents, as compared with the cells transfected with the NC mimic, based on IC50 data. Combination of miR-34a and chemotherapeutic agents inhibited Bcl-2 protein more significantly when compared with blank group,miR-34a group,and chemotherapeutic agents group.1. miR-126 inhibited migration and invasion of glioma cells,which changed their phenotype,perhaps partially by regulating Crk.2. miR-34a acted as a tumor suppressor in p53-mutant glioma cells U251, partially through regulating SIRT1.3.miR-34a modulated chemosensitivity of U251 glioma cells to chemotherapeutic agents by regulating the expressions of Bcl-2 protein.