Microrna-128 In Glioma In The Biological Function Of Research

MicroRNAs (miRNAs) are a class of small noncoding RNAs. They promote or repress gene expression by targeting the 3' untranslated regions (3'UTRs), 5' regions or protein coding regions. But the most familiar way until now is that they lead to translational silencing of the targets by combing to their 3'UTRs imperfectly. MiRNAs function in various process including physiology and pathology. Recent evidence indicates that miRNAs play important roles in tumor formation, and they also involved in glioblastomas which represent the most common malignant primary brain tumors.Mircroarray data showed many miRNAs expressed abnormally in glioblastoma, and among them, the brain-specific miR-128 is one of the most obviously down-regulated miRNAs. To study the function of miR-128 in glioblastoma, we first used the method of real-time PCR to verify the expression level of it in nine glioblastoma samples which contain grade II to grade IV glioblastoma three cases respectively, four glioblastoma cell lines (T98G, U87, U251 and A172) and two cases normal brain tissues. The results showed that miR-128 was down-regulated in glioblastoma tissues and cell lines compared to normal brain tissues, and the expression level is related with grade of glioblstoma, higher grade tumors have the lower expression of mir-128.To examine the role of miR-128 in glioblastoma cells, mature miR-128 was synthesized and transduced into T98G cell. Real-time PCR result showed the transfection was effective. After transfection of 48 hours, both of the MTT and BrdU experiments showed a reduction of cell proliferation. However, there was no apoptosis by hochest staining. So miR-128 might infect expression of some genes related with proliferation but not apoptosis. Combined with bioinformatic prediction, we chose eight putative target genes related with cell proliferation or neural differentiation which include E2F3a,BMI-1,SOX11 and so on, and did the luciferase assay in 293ET cells. The results showed that miR-128 could reduce the luciferase activity cloned with binding site of E2F3a. However, when the binding site was mutated, this kind of reduction was disappeared. Meanwhile, after transfection of miR-128 into T98G cells, the endogenous protein level of E2F3a was reduced without the change of mRNA level. These results showed miR-128 might target E2F3a.E2F3a is a transcription factor has an established role in controlling cell cycle progressing from G1 phase to S phase. The high expression of E2F3a can promote proliferation of cells while its downregulation can inhibit their proliferation. In our study, we also detected the expression levels of E2F3a in nine cases of glioblastoma samples, four glioblastoma cell lines and two cases of normal brain tissues by the methods of Western Blot and immunochemistry. All the results showed that E2F3a was obviously upregulated in glioblastoma, which was opposite to the expression levels of miR-128. What's more, reducing the expression level of E2F3a in a siRNA-mediated way could inhibit the proliferation of T98G cells, while overexpressing of E2F3a could rescue part of the affection induced by miR-128. All these results confirmed that miR-128 may affect the expression of E2F3a, and it also showed that miR-128 had other target genes in glioblastoma except E2F3a.In order to detect global impact on protein output, we used technology named pulse SILAC (stable isotope labelling with amino acids in cell culture, pSILAC) to detect the widespread affect of miR-128 on T98G. More than 1700 proteins were identified in mass, while 4 of them were up regulated more than 0.5 fold and 57 of them were down regulated more than 50%. Among these 57 genes, 7 of them including TROVE2, CCNK and so on had targets sites of miR-128 in their 3'UTR. They might be the direct target genes of miR-128a. However, other genes might have different binding aways or they might be the indirect function genes of miR-128.Concluded from our study, miR-128 was down regulated in glioblastoma cells and tissues, and it could inhibit the proliferation of glioblastoma through negatively targeting the genes related with proliferation. E2F3a is one these target genes. Besides that, miR-128 also inhibited expression of many other genes including TROVE2,CCNK and so on. However, their mechanisms still need to be studied.