miR-543对胶质瘤细胞SHG-44增殖和凋亡的影响

The Effect of miR-543 on Proliferation and Apoptosis in Glioma SHG-44 Cells

目的:观察miR-543对人胶质瘤细胞SHG-44增殖和凋亡的作用。方法:设计并合成miR-543寡核苷酸模拟物(miR-543 mimic)及阴性对照mimic NC序列、寡核苷酸抑制物(miR-543 inhibitor)及阴性对照inhibitor NC序列,将人胶质瘤细胞SHG-44株分为Control(对照组)、mimic NC组、inhibitor NC组、miR-543 mimic组和miR-543 inhibitor组,各组细胞转染相应序列; Real-time PCR检测miR-543及其靶基因氨酰基-tRNA合成酶相互作用多功能蛋白3(AIMP3) mRNA的表达,应用噻唑蓝细胞增殖实验(MTT)评估SHG-44细胞的增殖,应用流式细胞术检测细胞周期和细胞凋亡,应用Western Blot检测细胞周期蛋白cyclin D1、cyclin E与细胞凋亡相关蛋白Bcl-2、bax的表达变化。结果:与对照组相比,转染miR-543 mimic后,SHG-44细胞中miR-543 RNA表达水平显著上调(P <0. 01),其靶基因AIMP3 mRNA表达水平显著下调(P <0. 01);转染miR-543 inhibitor后,细胞中miR-543 RNA表达水平显著下调(P <0. 01),AIMP3 mRNA表达水平显著上调(P <0. 01);转染miR-543 mimic后,细胞增殖水平显著降低(P <0. 01);转染miR-543 inhibitor后,细胞增殖水平显著升高(P <0. 01);转染miR-543 mimic后,细胞处于G1期的细胞数量明显增加(P <0. 01),同时细胞凋亡率也显著增加(P <0. 01),而转染miR-543 inhibitor后细胞凋亡率显著减少(P <0. 01); Westrn Blot结果显示,与对照组相比,转染miR-543 mimic后,细胞周期蛋白cyclin D1、cyclin E及抗凋亡相关基因Bcl-2蛋白表达显著下调(P <0. 01),促凋亡相关基因bax蛋白表达显著上调(P <0. 01);转染miR-543 inhibitor后,细胞中cyclin D1、cyclin E、Bcl-2蛋白表达显著上调(P<0. 01),bax蛋白表达显著下调(P <0. 01)。结论:miR-543可能通过下调细胞周期蛋白cyclin D1、cyclin E及抗凋亡相关基因Bcl-2的表达,上调促凋亡基因bax的表达进而抑制细胞增殖,促进细胞凋亡。

Objective:To observe the role of miR-543on the proliferation and apoptosis of human glioma SHG-44cells and to investigate its effect on the protein expression of cyclin D1,cyclin E,Bcl-2and bax.Methods:miR-543oligonucleotide mimics(miR-543mimic)and oligonucleotide inhibitor(miR-543inhibitor)were used to transfect SHG-44cells.Real-time PCR was used to detect the expression of miR-543and AIMP3RNA;methylthiazol tetrazoliu(MTT)assay was used to evaluat cell proliferation;Flow cytometry was applied to detect cell cycles and apoptosis;The expression of cyclin D1,cyclin E,Bcl-2and bax were detected by Western Blot.Results:Compared with the control group,the expression of miR-543in SHG-44cells was significantly up-regulated(P<0.01)and the expression of AIMP3mRNA was significantly down-regulated(P<0.01)after transfection of miR-543mimic;the expression of miR-543in SHG-44cells was significantly down-regulated(P<0.01)and that of AIMP3mRNA was significantly up-regulated(P<0.01)after transfection of miR-543inhibitor.The cell proliferation level significantly decreased after transfection of miR-543mimic(P<0.01),but after transfection of miR-543inhibitor,the cell proliferation level significantly increased;The number of cells in G1phase and also the apoptosis rate significantly increased(P<0.01)after transfection of miR-543mimic(P<0.01);but the apoptosis rate significantly decreased(P<0.01)after transfection of miR-543inhibitor.Compared with the control group,Western Blot results showed that the expression of cyclin D1,cyclin E and Bcl-2was significantly down-regulated(P<0.01),meanwhile the expression of bax protein was significantly up-regulated(P<0.01)after transfection of miR-543mimic,while the expression of cyclin D1,cyclin E and Bcl-2was significantly up-regulated(P<0.01)and the expression of bax protein was significantly down-regulated(P<0.01)after transfection of miR-543inhibitor,.Conclusion:miR-543can inhibit cell proliferation and promote apoptosis by lowering the cyclin D1,cyclin E and Bcl-2expression level meanwhile elevating bax expression level.