摘要
目的探讨微小RNA122(miR-122)对神经胶质瘤细胞凋亡的影响及可能分子机制。方法采用荧光定量PCR(QPCR)检测人正常星形胶质细胞系HA1800和胶质瘤细胞系U251、U87、SHG44、MO59K中miR-122的表达情况。双荧光素酶报告实验评价miR-122对RUNX2的靶向调控作用。向U251细胞转染miR-122 mimics(miR-122组)和阴性对照NC(NC组),流式细胞术检测细胞凋亡,Western blotting检测凋亡相关蛋白Bcl-2、Bax、cleaved caspase-3的表达。向U251细胞同时转染miR-122 mimics和siRUNX2(miR-122+siRUNX2组),流式细胞术检测细胞凋亡。结果 U251、U87、SHG44、MO59K细胞系中miR-122表达水平分别为0. 34±0. 052、0. 65±0. 061、0. 59±0. 071和0. 69±0. 098,显著低于正常星形胶质细胞系HA1800的1. 17±0. 173,差异具有统计学意义(P<0. 05)。miR-122可抑制野生型RUNX2 3’UTR报告基因载体的荧光素酶活性,而对突变型RUNX2 3’UTR-MUT的荧光素酶活性无影响。miR-122组U251细胞凋亡率为(23. 77±0. 83)%,高于NC组的(6. 17±0. 12)%,差异具有统计学意义(P<0. 05);低于miR-122+siRUNX2组的(70. 85±0. 35)%(P<0. 05)。Western blotting检测结果显示,miR-122组RUNX2蛋白表达量为0. 57±0. 048,低于NC组的1. 21±0. 19(P<0. 05)。与NC组比较,miR-122组Bax(3. 47±0. 077)、cleaved caspase-3(4. 38±0. 121)表达均明显上调,而Bcl-2(0. 39±0. 104)明显下调(P<0. 05)。结论 miR-122可以通过靶向RUNX2促进线粒体凋亡通路的激活,促进神经胶质瘤细胞凋亡。
Objective To investigate the effect of microRNA122(miR-122)on apoptosis of glioma cells and its possible molecular mechanism.Methods The expression of miR-122 in human normal astrocyte lines HA1800 and glioma cell lines U251,U87,SHG44 and MO59K was detected by quantitative fluorescence PCR(QPCR).Dual luciferase reporter assay was used to evaluate the targeting effect of miR-122 on RUNX2.MiR-122 mimics(miR-122 group)and negative control NC(NC group)were transfected into U251 cells.Apoptosis was detected by flow cytometry.Western blotting was used to detect the expression of Bcl-2,Bax and cleaved caspase-3 protein in two groups.MiR-122 mimics and siRUNX2(MiR-122+siRUNX2 group)were simultaneously transfected into U251 cells.Apoptosis was detected by flow cytometry.Results The expression of miR-122 in U251,U87,SHG44 and MO59K cell lines were 0.34±0.052,0.65±0.061,0.59±0.071 and 0.69±0.098,which was significantly lower than that of human normal astrocyte lines HA1800(1.17±0.173),and the difference was statistically significant(P<0.05).MiR-122 inhibited the luciferase activity of wild-type RUNX2 3'UTR reporter gene vector,but had no effect on the luciferase activity of mutant RUNX2 3'UTR reporter gene vector.The apoptotic rate of U251 cells in miR-122 group was(23.77±0.83)%,higher than that in NC group(6.17±0.12)%,lower than that in miR-122+siRUNX2 group(70.85±0.35)%.The difference was statistically significant(P<0.05).Western blotting results showed that the expression of RUNX2 protein in the miR-122 group was 0.57±0.048,which was lower than that in the NC group(1.21±0.19),with statistical significance(P<0.05).Compared with NC group,the expressions of Bax and cleaved caspase-3 were significantly up-regulated in miR-122 group,while Bcl-2 was significantly down-regulated(P<0.05).Conclusion MiR-122 can promote the activation of mitochondrial apoptosis pathway by targeting RUNX2,thereby promoting apoptosis of glioma cells.
作者
杨才弟
王丽娟
曾鼎华
朱剑梅
YANG Caidi;WANG Lijuan;ZENG Dinghua;ZHU Jianmei(Department of Neurology,Sichuan People s Hospital,Sichuan Academy of Medical Sciences,Chengdu 610100,China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2019年第2期124-128,共5页
Chinese Clinical Oncology
