miR-377过表达对胶质母细胞瘤细胞增殖、凋亡、迁移的影响及机制

Effect of miR-377 overexpression on cell proliferation,apoptosis,and migration of glioblastoma

目的探讨微小RNA-377(miRNA-377)过表达对胶质母细胞瘤细胞增殖、凋亡、迁移的影响,并探讨其作用机制。方法常规培养胶质母细胞瘤细胞系A172、U373、U87、U251及正常脑胶质细胞系HEB。用qRTPCR法检测细胞中miR-377的表达。将U251细胞随机分为miR-377组、miR-CON组及Blank组,分别转染miR-377mimics、Scramble及空白对照,用qRT-PCR法检测转染效率。用MTT法检测细胞增殖能力,流式细胞术测定细胞凋亡能力,用细胞划痕实验检测细胞迁移能力。用Western blotting法检测细胞内组蛋白去乙酰化酶9(HDAC9)和E26转录因子1(ETS-1)蛋白表达。结果转染24 h,miR-377组miR-377相对表达量高于miR-CON组、Blank组(P均<0. 05),转染成功。与miR-CON组、Blank组比较,转染72、96 h miR-377组细胞增殖能力低(P均<0. 05),细胞凋亡率高(P均<0. 05),细胞划痕愈合率低(P均<0. 05)。miR-377组HDAC9、ETS-1蛋白相对表达量均低于miRCON组、Blank组(P均<0. 05)。结论 miR-377过表达可抑制胶质母细胞瘤细胞增殖、迁移,并促进其凋亡,其机制可能与下调HDAC9、ETS-1蛋白表达有关。

Objective To investigate the effect of miR-377 overexpression on cell proliferation,apoptosis,and migration of glioblastoma and its mechanism.Methods The expression of miRNA-377 was detected in the conventional cultured glioblastoma cell lines A172,U373,U87,U251 and normal glioblastoma cell line HEB by qRT-PCR,which confirmed that the expression of miRNA-377 in glioblastoma cell lines was low,and with the lowest in U251 cells.The glioma cell line U251 was divided into the miR-377 group,miR-CON group,and Blank group,which were transfect with miR-377 mimics,Scramble,and PBST,respectively.Transfection efficiency was measured by qRT-PCR.MTT assay was used to measure the proliferation ability.Flow cytometry was used to measure the apoptosis ability.Cell scratch test was used to measure the migration ability.The expression of histone deacetylase 9(HDAC9)and E2 6 transformation-specific-1(ETS-1)protein was measure by Western blotting.Results The expression of miR-377 in the glioblastoma cell lines A172,U373,U87,and U251 was significantly lower than that in HEB cells(P<0.05),with the lowest in U251 cells.After 24 hours of transfection,the relative expression of miRNA-377 in the miRNA-377 group was higher than that in the miRNA-CON group and the Blank group(P<0.05),which showed the transfection was successful.At 72 and 96 h,the OD value of the miR-377 group was significantly lower than that in the miR-CON group(P<0.05).The apoptosis rate of the miR-377 group was significantly higher than that in the miR-CON group(P<0.05).The healing rate of scratches in the miR-377 group was significantly lower than that in the miR-CON group(P<0.05).The expression levels of HDAC9 and ETS-1 protein in the miR-377 group were significantly lower than those in the miR-CON group and Blank group(both P<0.05).Conclusion The miR-377 can inhibit the proliferation and migration,and promote apoptosis of glioma cells,and its mechanism may be associated with down-regulated expression of HDAC9 and ETS-1.