miR-508-5p介导mTOR对骨肉瘤细胞自噬的影响

The Effect of mTOR on Autophagy of Osteosarcoma Cells Mediated by miR-508-5p

目的:探讨miR-508-5p对骨肉瘤细胞自噬及雷帕霉素靶向基因(mTOR)信号通路的影响。方法:收集近五年收治的59例骨肉瘤患者的癌组织与癌旁正常组织,另购买人骨肉瘤细胞株MG-63与人成骨细胞hFOB,采用RT-PCR法检测各组织与细胞中miR-508-5p与mTOR mRNA的相对表达量;将MG-63细胞分成三组:空白组不进行转染,NC组转染空载质粒,过表达组转染miR-508-5p重组质粒,采用CCK-8法检测转染培养后的三组细胞增殖情况,流式细胞仪检测三组细胞凋亡率,采用RT-PCR法检测各组miR-508-5p与mTOR mRNA的相对表达量;采用Western blot法检测细胞中mTOR、Bax、Bcl-2、LC3-Ⅰ与LC3-Ⅱ蛋白的相对表达量。结果:相比癌旁正常组织与hFOB细胞,癌组织与MG-63细胞中miR-508-5p相对表达量均明显降低,而mTOR mRNA的相对表达量均明显升高,差异均有统计学意义(P<0.05);相比空白组与NC组,过表达组miR-508-5p相对表达量明显上升,mTOR mRNA的相对表达量明显降低,细胞OD值明显降低,凋亡率明显增加,Bax与LC3-Ⅱ蛋白表达量均升高,而mTOR、Bcl-2与LC3-Ⅰ蛋白相对表达量均降低,差异均有统计学意义(P<0.05)。结论:miR-508-5p在骨肉瘤细胞中呈现低表达趋势,过表达miR-508-5p后可通过抑制mTOR通路,引发细胞自噬,从而抑制细胞增殖与促进细胞凋亡。

Objective:To investigate the effect of miR-508-5p on autophagy and rapamycin targeting gene(mTOR)signaling pathway in osteosarcoma cells.Method:A total of 59 cases of osteosarcoma in our hospital in the past five years were collected from cancer tissues and adjacent tissues,and human osteosarcoma cell line MG-63 and human osteoblasts hFOB were purchased.miR-508-5p and mTOR were detected by RT-PCR MG-63 cells were divided into three groups:blank group was not transfected,NC group was transfected with empty plasmid,over expression group was transfected with miR-508-5p recombinant plasmid,CCK-8 method was used to detect the proliferation of three groups of cells after transfection,flow cytometry was used to detect the apoptosis rate of three groups,RT-PCR method was used to detect the relative expression of miR-508-5p and mTOR mRNA in each group;Western blot was used to detect the relative expression of mTOR,Bax,Bcl-2,LC3-Ⅰand LC3-Ⅱin the cells.Result:The relative expression of miR-508-5p in cancer tissue and MG-63 cell were significantly lower than those in normal tissue and hFOB cell,while the relative expression of mTOR mRNA were significantly higher,the differences were statistically significant(P<0.05).Compared with the blank group and NC group,the relative expression of miR-508-5p,mTOR mRNA,odvalue,apoptosis rate,Bax and LC3-Ⅱprotein in the overexpression group increased significantly,while the relative expression of mTOR,Bcl-2 and LC3-Ⅰprotein decreased significantly,the differences were statistically significant(P<0.05).Conclusion:The expression of miR-508-5p was low in osteosarcoma cells,and the over expression of miR-508-5p can induce autophagy by inhibiting mTOR pathway,thus inhibiting cell proliferation and promoting cell apoptosis.