Mir - 214 In Ubc9 Expression Regulation And Its Influence On Glioma Cell Apoptosis

Small ubiquitin-like modifier(SUMO), is a member of ubiquitin and ubiquitin-like superfamily. Similar to ubiquitination, sumoylation requires three steps of enzymatic reactions to form colavent bond between the C-terminal of SUMO protein and the lysine residue of substrate. Sumoylation regulates various process in physiology and pathology. Recent evidence indicates that sumoylation involves in tumor formation.Ubiquitin-like protein SUMO conjugating enzyme9(Ubc9) is the sole E2-conjugating enzyme essential for sumoylation, which is up-regulated in an increasing number of human cancers, such as ovarian carcinoma and lung cancer.To study the function of Ubc9in glioblastoma, we first used Western blot to verify the expression level of it in nine glioblastoma tissue samples including three cases respectively from grade Ⅱ to grade Ⅳ, four glioblastoma cell lines (T98G, A172, U87MG and U251) and two cases of normal brain tissues. The results showed that Ubc9was up-regulated in glioblastoma tissues(grade Ⅲ and grade Ⅳ) and three cell lines(T98G, A172and U251) compared to normal brain tissues, and the expression level is related with grade of glioblstoma, higher grade tumors with the higher expression of Ubc9. Real-time PCR results showed that the mRNA level of Ubc9didn’t match Ubc9protein level in four glioblastoma cell lines, suggesting the existence of a post-transcriptional regulatory mechanism in charge of Ubc9protein expression, microRNA(miRNA) maybe involve in the expression regulation of Ubc9in glioblastoma cells.MiRNAs are a class of small noncoding RNAs. They usually lead to translational silencing of the targets by binding to their3’UTRs imperfectly. miRNAs function in various physiology and pathology process. Recent evidences indicate that miRNAs play important roles in tumor formation.Bioinformatics analysis and biological experiments were used to find the potential miRNAs targeting Ubc9. Firstly, we used TargetScan to analyze Ubc93’UTR for predicting the miRNAs involved in this biological process, then verified the expression level of those miRNAs in glioblastoma samples and cell lines by real-time PCR. Since Ubc9is up-regulated in glioblastoma samples and cell lines compared to normal brain tissues, we chose the miRNAs down-regulated in glioblastoma. Dual luciferase activity assay results showed that miR-214could reduce the luciferase activity cloned with the3’UTR of Ubc9. Furthermore, mutation of the binding site abolished this effect. To further verify the role of miR-214in regulation of endogenous Ubc9in glioblastoma, mature miR-214was synthesized and introduced into T98G and A172cells. We found that enforced expression of miR-214could inhibit the proliferation of both cell lines. TUNEL assay indicated that enforced miR-214significantly induced apoptosis in both cell lines. Meanwhile, after transfection of miR-214into T98G and A172cells, the endogenous protein level of Ubc9was reduced. These results showed that miR-214might target Ubc9.To examine the role of Ubc9in glioblastoma cells, specific siRNA of Ubc9was synthesized and transfected into T98G and A172cell lines. After transfection for48hours, MTT experiment showed a reduction of cell proliferation. The results of TUNEL assay was similar to that induced by enforced expression of miR-214. All of these results confirmed that miR-214may affect the expression of Ubc9. Western blot results indicated that the reduction of Ubc9protein level accompany with the lower sumoylation of proteins in T98G cells.In conclusion, miR-214is down-regulated in glioblastoma cell lines and tissues, which release the inhibition of Ubc9expression and changes the sumoylation status in glioblastoma cells, furthermore, this change induces the dysregulation of apoptosis and proliferation.