miR-18a对胶质瘤细胞增殖和自噬的影响

Effects of miR-18a on proliferation and autophagy of glioma cells

目的:探讨miR-18a在神经胶质瘤细胞增殖和自噬中的作用。方法:采用瞬时转染的方法将miR-18a inhibitor及其阴性对照(negative control inhibitor, NC inhibitor)分别转染大鼠C6胶质瘤细胞;实时荧光定量聚合酶链反应(RT-qPCR)检测miR-18a在C6胶质瘤细胞中的表达;CCK-8法检测转染miR-18a inhibitor后C6细胞的增殖活性;mCherry-EGFP-LC3B质粒瞬时转染各组细胞,以动态监测自噬通量;Western blot检测敲低miR-18a后C6细胞中自噬相关蛋白LC3、p62、Beclin1的表达情况;RT-qPCR检测转染miR-18a抑制剂后C6细胞中自噬相关基因Beclin1和LC3B的相对表达水平。结果:RT-qPCR结果显示,与NC inhibitor组相比,miR-18a inhibitor组的miR-18a表达量显著降低(P<0.05)。敲低miR-18a后显著抑制C6胶质瘤细胞的增殖(P<0.05)。倒置荧光显微镜下观察到抑制miR-18a后可阻断自噬流,抑制自噬小体和溶酶体融合。抑制miR-18a可使胶质瘤细胞中LC3-Ⅱ/LC3-Ⅰ和Beclin1蛋白的相对表达量显著降低(P<0.05),p62蛋白的相对表达量明显升高(P<0.05)。敲低miR-18a后能抑制胶质瘤细胞中的Beclin1和LC3B mRNA表达(P<0.05)。结论:miR-18a下调可抑制胶质瘤细胞的增殖和自噬。

Objective:To investigate the role of miR-18 a in the proliferation and autophagy of glioma cells.Methods:C6 cells were transfected with miR-18 a inhibitor and its negative control inhibitor(NC inhibitor) respectively by transient transfection method.The expression of miR-18 a in C6 glioma cells was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The proliferation activity of C6 cells after transfection with miR-18 a inhibitor was detected by CCK-8 assay.Autophagy flux was monitored by transfecting mCherry-EGFP-LC3 B plasmid.Western blot was used to detect the expression level of autophagy-related proteins LC3,p62 and Beclin1 in C6 cells transfected with miR-18 a inhibitor.RT-qPCR was used to detect the relative expression level of autophagy-related genes Beclin1 and LC3 B in C6 cells transfected with miR-18 a inhibitor.Results:The results of RT-qPCR showed that the expression level of miR-18 a in glioma cells transfected with the miR-18 a inhibitor was significantly decreased compared with NC inhibitor(P<0.05).Knockdown of miR-18 a significantly inhibited the proliferation of C6 glioma cells(P<0.05).It was observed under fluorescence microscope that inhibition of miR-18 a could block the autophagy flow and inhibit the fusion of autophagosome and lysosome.Inhibition of miR-18 a significantly decreased the relative expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin1 proteins in glioma cells(P<0.05),and significantly increased the relative expression level of p62(P<0.05).Beclin1 and LC3 B mRNA expressions were inhibited after miR-18 a knockdown in glioma cells(P<0.05).Conclusion:Down-regulation of miR-18 a can inhibit the proliferation and autophagy of glioma cells.