Study On The Tumor Suppressive Effects Of MiR-218on Gliomas And The Mechanisms

Objectives:microRNA-218(miR-218) is an important tumor-suppressive microRNA (miRNA) whose expression level decreased abnormally in multiple solid tumors and precancerous lesions. Our previous study showed that its expression level decreased along with the elevation of the malignant degree of gliomas and suggested it played a pivot role in the pathogenesis and progression of gliomas. Bioinformatics predicts cyclin-dependent kinase6(CDK6) a potential target of miR-218, and this hypothesis was strengthened greatly by the negative correlation between their expression levels in gliomas of various grades. But the real relationship between these two molecules in glioma cells is still to be verified. The aim of the current study is to observe the tumor suppressive effects of this miRNA on the proliferation, apoptosis, migration, invasion and differentiation of malignant glioma cells, verify whether CDK6is a natural target of miR-218, and explore whether miR-218exerts its tumor suppressive effects by specifically blocking the expression of CDK6.Methods:①The miR-218overexpressing sub-cell lines of U87MG and SNB-19and their control sub-cell lines were established by plasmid transfection and G418screening and their miR-218expression levels were compared by stem-loop quantitative RT-PCR (qRT-PCR).②Luciferase assay, qRT-PCR and Western blot were used to demonstrated whether CDK6is the natural target of miR-218and how miR-218down-regulates the expression of CDK6.③The proliferative activity of the glioblastoma sub-cell lines were compared by Ki-67immunocytochemistry and in vitro colony formation assay, and the cell cycle distribution were analyzed by flow cytometry (FCM) and the immunoblotting of cyclin D1, cyclin E, cyclin A, CDK4and CDK6.④The apoptotic levels of the miR-218overexpressing sub-cell lines and the miR-218mimics transfected tumor cells were assessed by Caspase3/7assay and FCM, respectively. The influence of CDK6on apoptosis and the role of its downregulation in the pro-apoptotic effects of miR-218were clarified by introducing the CDK6expressing plasmid to the sub-cell lines and the cells with high levels of miR-218.⑤The effects of miR-218on the migratory and invasive abilities of the glioblastoma cells were assessed by in vitro migration and invasion assay as well as matrigel3D culture of the4sub-cell lines and the quantitative invasion assay of the miR-218mimics and nonscrambled control sequence transfected tumor cells. Then the cells with high miR-218levels were compensated with exogenous CDK6to demonstrate whether miR-218exerted the inhibitory effects by downregulating CDK6.⑥The expression levels of CD133, Nestin and glial fibrillary acidic protein (GFAP) in the4glioblastoma sub-cell lines with or without exogenous CDK6were assessed by Western blot and immunocytochemistry to find out whether miR-218could induce the maturation and end-differentiation of the malignant glioma cells and the glioma stem cells and whether these effects were mediated by CDK6downregulation. The specificity of these modulating effects was further validated by immunofluorescence against CD133and Nestin.Results:①The miR-218expression levels of the newly established U87MG and SNB-19miR-218overexpressing sub-cell lines were11.58±2.39and26.23±8.08folds of those of their corresponding controls.②Luciferase assay showed that miR-218could effectively block the expression of the luciferase gene linked to the coding region of CDK63’UTR and demonstrated that CDK6was the natural target of miR-218. qRT-PCR and Western blot showed dramatic decrease of the expression levels of CDK6mRNA and protein in the miR-218overexpressing sub-cell lines.③The Ki-67labeling index (LI) and the expression levels of cyclin E, cyclin A were significantly lower in the miR-218overexpressing sub-cell lines than in their corresponding controls, while the expression levels of cyclin D1were significantly higher. However, the expression of CDK4remained unchanged. FCM showed prominent Gi arrest in the miR-218overexpressing sub-cell lines.④The miR-218overexpressing sub-cell lines showed significantly higher Caspase3/7bioactivity while the miR-218mimics transfected U87MG and SNB-19cells showed higher apoptotic indexes. Exogenous CDK6failed to lower the elevated Caspase3/7activity and the increased apoptotic index.⑤In vitro migration and invasion assays,3D cultivation and quantitative invasion assays all showed that miR-218could effectively undermine the migratory and invasive abilities of the malignant glioma cells, while exogenous CDK6could partially reverse these inhibitory effects.⑥The expression levels of CD133and Nestin of the miR-218overexpressing sub-cell lines were significantly lower than those of the control sub-cell lines, while the GFAP LI of the former was significantly higher than the latter. Exogenous CDK6could partially reverse the above modulating effects of miR-218. Immunofluorescence showed that the downregulation of CD133and Nestin took place specifically in the cells successfully transfected by the miR-218expression plasmid and expressing the EGFP reporter.Conclusions:①In the current research, we have successfully established the glioblastoma sub-cell lines that can stably overexpressing miR-218.②CDK6is the natural target of miR-218. miR-218can downregulate the expression of CDK6by blocking the translation and inducing the degradation of its mRNA.③miR-218can induce G1arrest and inhibit the proliferation of the malignant glioma cells by blocking CDK6expression.④miR-218can induce the apoptosis of the malignant glioma cells, but CDK6downregulation is not involved in this process.⑤miR-218can effectively undermine the migratory and invasive abilities of the glioblastoma cells, and CDK6downregulation is one of the molecular pathway mediating this inhibitive effect.⑥miR-218can induce the maturation and end-differentiation of the malignant glioma cells and the glioma stem cells and inhibit the phenotype of the progenitor cells. This effect is achieved at least partly by knocking down the expression of CDK6.