Study On The Mechanism Of MicroRNA-26b And MicroRNA-125b Regulate Glioma Cell Proliferation And Apoptosis

microRNAs(miRNAs) are a kind of short single stranded RNA molecules, which usually have ~22nt long and highly conserved in evolution. They regulate gene expression throughsequence specific interactions with target mRNAs resulting in translational repression or mRNA degradation, such regulating gene expression in the post-transcription level. The abnormal levels of miRNAs in tumors have important pathogenetic consequences. Many miRNAs are reported have great function on tumorigenesis and tumor development. Glioma is the most common malignant tumor in central nervous system tumors. Recent researches have shown that miRNAs have great relationship with glioma development.We found miR-26b was down-regulates in giloma cells, but the real function of miR-26b in glioma cells was not been notice yet. In the present work we do the research work on the role of miR-26b on glioma development and try to find its target genes in glioma cell. miR-125b is found expressed with a high level in embryo nervous system development, while its expression level will turn to a relatively low level in brain tissues after born or grown up. The recent research found miR-125b expressed in glioblastoma abnormally, however, the function of miR-125b in glioblastoma and its role on regulation glioma cell proliferation and apoptosis were not been knew well. In the present study we investigate the expression characteristics of miR-125b in different WHO grade glioma tissues and its role on regulates glioma cell proliferation, apoptosis, the effects on anti-tumor drug and the way of miR-125b regulates glioma cell apoptosis. Recent work point that miRNAs take a great role in tumor stem cell development, but the role of miRNAs on brain tumor stem cell was not known. In this work we will found the different miRNA expression profile between brain tumor cell and glioma cell and try to find the role of miR-125b on brain tumor cell proliferation and differentiation.We frist detect the expression level of miR-26b and miR-125b in normal brain tissues and different WHO grade glioma tissues and in human U87 MG, U251glioma cell lines and rate C6 glioma cell line by Real-time PCR. The results show miR-26b was down-regulated with the increased grade of glioma, menwhile its expression level was relatively low in the three glioma cell lines. Over expression of miR-26b by transfect miR-26b mimics to U251 and C6 cells, resulted glioma cell proliferation, migration and invasion ability were inhibited. We then tried to find the target genes of miR-26b using the online miRNA target prediction programs. The prediction results show Epha2 is a potential target gene of miR-26b. Here we detected the expression of EphA2 in normal brain, glioma samples of different grades, and three glioma cell lines and found that expression of EphA2 was significantly enhanced with the advance of glioma grade(p< 0.01), accompanying the decrease of miR-26b. Luciferase reporter experiment show miR-26b could specifically binding to the EphA2 3'UTR to inhibit the luciferase expression level. Western blotting expreiments confirmed that over-expression miR-26b inhibit the endogenous EphA2 protein level. Moreover, the glioma cell vasculogenic mimicry (VM) formation experiments show that miR-26b could specifically down-regulation EphA2 expression level to inhibit glioma cell VM network formation. In the U87MG cells which have very low level of EphA2 expression, the effects of miR-26b on cell proliferation, migration and invasion were not as notable as in EphA2 high expressed glioma cell. The results indicate that miR-26b regulates glioma cell is correlated with the EphA2 expression level.We found that miR-125b is up-regulated with the increased grade of glioma, and its expression level was relatively high in the three glioma cell lines. Over-expression miR-125b promote glioma cell proliferation and cell cycle anlaysis show that the S stage of glioma cell cycle was increased. Down-regulated miR-125b expression by transfected 2'methoxy modified miR-125b antisense into glioma cells the cells proliferation were inhibited. Hoest33342 stain, AV-FITC flow cytometer apoptosis anlaysis and TUNEL expriments show that knock down of miR-125b increase the glioma cell apoptosis. We try to investigated whether miR-125b mediated chemoresistance to chemotherapeutics agent ACNU in glioblastoma cells. miR-125b was cooperated with ACNU, and the results show over-expression miR-125b inhibit the U87MG cell sensitivity to ACNU, however, when knock-down of miR-125b inU87MG cells significantly enhance the cell sensitivity to ACNU. The results indicate that the expression of miR-125b in glioma cell could suppress the chemo-sensitivity of glioblastoma. 3) P53 wild type glioma cell (U87) and p53 mutated glioma cell (U251) and a p53 knocked-down U87 cells, which stable expression of miR-125b or miR-125b knocked down, were used to investigate the miR-125b affects glioma cell apoptosis if in a p53 dependent way. Western blotting was used to detect the protein expression level of p53, BCL-2, BAX, Caspase-9 and Caspase-3. The results show over-expression miR-125b inhibites the p53 expression level and knock-down of miR-125b in U87 MG cells p53 and caspase-3 were obviously activated. However, in p53 mutated glioma cell (U251) and p53 knocked-down U87 cells, the cell apoptosis induced by down-regulation of miR-125b were still happen, which indicated miR-125b regulation glioma cell apoptosis is not in a p53 dependent way. Further research found that p38MAPK protein was specific activated in miR-125b knocked down cells. When a p38MAPK specific inhibitor SB203580 added into U87 cell, the cell apoptosis induced by the miR-125b down-regulated have not significant change. The results indicated that miR-125b regulated glioma cell apoptosis in a p53 independent and p38MAPK independent way.The brain stem-like cells (BSC) cells were isolated from malignant glioma cell line U87 MG and the microarray detection system was employed to analyze whether there was difference in between BSC cells and U87 MG cells. Compared with U87 MG cells, 30 microRNAs were up-regulated with more than 5 fold in BCSCs than in U87 MG parent cells, in which miR-125b was up-regulated 7.56 fold. Overexpression miR-125b in BSC cells inhibited cell differentiation. Our study suggested that miRNAs may participate in regulating GSC cells growth and differentiation, and miR-125b plays a key role in regulating BSCs proliferation and differentiation.In conclusion, Our present research demonstrated that miR-26b may act as a tumor suppressor in glioma and it directly regulates EphA2 expression in glioma cell. miR-125b expressed in glioma cell could promote cell proliferation, inhibits cell apoptosis and suppress the ACNU chemo-sensitivity of glioblastoma. miR-125b regulated glioma cell apoptosis in a p53 independent and p38MAPK independent way. We also discovered that BSCs possessed a unique miRNA profile from U87MG cell, and the different expressed miRNAs may participate in regulating BSC cells growth and differentiation, in which miR-125b plays a key role in regulating BSCs proliferation and differentiation.