Relationship Between The Expression Levels Of MiR-218 And CDK6 As Well As Their Impacts On Tumor Cell Proliferation And Apoptosis In Gliomas

Objectives:microRNA(miRNA) is a non-coding single strand RNA, which can degrade mRNA or mediate translational repression of the target transcript by binding with the complementary seqence of target mRNA, then silence the expression of target mRNA by the above mechanism. miRNA regulates the expression of many oncogenes or tumor suppressor genes, and plays a important role in the development and progression of tumors.The abberant down-regulated expression of miR-218 had been observed in several malignant tumors. The up-regulation of target mRNA caused by the aberrantly decreased miR-218 expression could serve as a crucial factor just like tumor suppressor genes in regulating the proliferation, apoptosis, angiogenesis, invation and metastasis of tumors. Bioinformatics analysis showed that the mRNA of Cyclin-dependent kinase (CDK6) was the target mRNA of miR-218. CDK6 is an important regulator of cell cycle, which can promote the cell cycle from G1 to S phase. However, the expression of miR-218 and CDK6, their relationship in glioma cells as well as their effect on the proliferation and apoptosis of glioma cells have not been understanding at present. The object of this study would want to investigate above-mentioned questions.Methods:①The expression levels of miR-218, CDK6 and Ki-67 in 60 cases of different grades of gliomas and 10 control brain tissues were detected by means of tissue microarray, locked oligonucleotide probe in situ hybridization and immunohistochemistry, whilst the relationships among these results were analyzed.②malignant glioma cell line U87MG was divided into two groups, named as control group(thansfected with miRNA Negative Control) and mimics group(thansfected with miR-218 mimics).③qRT-PCR was adopted to measure the relative level of miR-218 in the two groups.④The expression levels of CDK6 and Ki-67 in the two groups were evaluated using immunocytochemistry.⑤The apoptotic alterations in the two groups were detected by single cell gel electrophoresis.⑥The effect of miR-218 on the rumor cell growth was measured by MTT.Results:①The miR-218 labeling indexes (LI%) of the control group and glioma groups of gradeⅠ～Ⅱ,Ⅲand IV were 22.45±0.594.00±1.07,1.87±1.06 and 0.94±0.78, respectively; there were statistically significant all of differences among the four groups(P<0.05-0.001).②The detection of immunohistochemistry demonstrated that CDK6 LI% of the above four groups were 7.25±1.20,16.71±0.80,24.43±0.62 and 32.05±0.43, respectively. There were significant differences between the control group and each of the glioma groups as well as the gradeⅠ～Ⅱand grade IV glioma groups (P<0.01-0.001). Ki-67 positive cell densities of the four groups were 0.00±0.00, 9.30±3.48,31.15±9.44 and 60.15±13.60, respectively. All of the differences of the four groups had statistical significance among one another (P<0.01).③miR-218 LI% negatively correlated with CDK-6 LI%(r=-0.480, P<0.01) and Ki-67 positive cell density (r=-0.534, P<0.01), while the latter two positively correlated with each other (r= 0.530, P＜0.01).④The results of qRT-PCR confirmed that miR-218 level in U87MG cells thansfected with mimics was significantly higher than that of control group (P＜0.001). The CDK6 LI%(14.74±1.19) and Ki-67 LI%(30.88±3.31) of the mimics group were significantly lower than those (79.06±2.07 and 64.94±3.96, respectively) of the control group (P<0.001).⑤The analysis of single cell gel electrophoresis manifested that apoptotic indexes (AI%) of the mimics group and control group were 68.44±7.05 and 13.04±0.97, respectively, which were significantly different from each other (P<0.001).⑥The value of relative absorbances of the mimics group and control group at 24h,48h,72h and 96h after transfection were 0.64±0.02,0.72±0.08,1.35±0.28, 1.63±0.14 and 0.67±0.06,1.23±0.14,1.91±0.04,2.19±0.22, respectively. Significant difference existed between the two groups at other time points besides at the 24h (P＜0.05～0.001).Conclusions:①There are universally the decrease of miR-218 expression and increase of CDK6 expression in glioma cells. The expression levels of them were the important parameters on the assessment of the benign and malignant degrees of gliomas.②That aberrantly decrease of miR-218 expression results in the up-regulation of CDK6 is probably a crucial factor promoting the proliferation of tumor cells.③The supplement of the engeneous miR-218 may effectively down-regulate CDK6 expression in the tumor cells, inhibit its proliferation and promote its apoptosis. Therefore, miR-218 has the important potential practicability on the genetherapy of malignant gliomas.