H_2O_2诱导的HT-29结肠癌细胞凋亡中microRNA-23表达变化

MicroRNA-23 was involved in H_2O_2-induced apoptosis of HT-29 cells

目的观察过氧化氢(H2O2)诱导的HT-29结肠癌细胞凋亡中microRNA(miRNA)的表达变化及其在细胞凋亡中的作用。方法常规培养的HT-29结肠癌细胞,给予500 nM H2O2处理48 h,构建HT-29细胞凋亡模型;常规RNA抽提后,利用miRNA颈环结构引物进行反转录反应后,采用实时荧光定量PCR分析miRNAs的表达水平。构建miR-23的Psuper过表达质粒,并将其转染HT-29细胞。实时荧光定量PCR分析Psuper-miR-23在HT-29细胞中的过表达效率。在H2O2诱导的HT-29细胞中过表达miR-23,48 h后通过流式细胞术检测HT-29细胞的凋亡情况,并通过定量PCR检测细胞中Caspase 3的表达。结果在H2O2诱导的HT-29结肠癌细胞凋亡模型中,miR-22、miR-24、miR-26、miR-30、miR-181和miR-214的表达水平在对照组与H2O2组之间表达差异没有显著性,而miR-23在处理组的表达水平明显升高。Psuper-miR-23过表达质粒转染培养的HT-29细胞能够明显提高miR-23的表达水平。在H2O2诱导的HT-29细胞过表达miR-23,能够促进HT-29细胞的凋亡。结论 miR-23在H2O2诱导的HT-29细胞凋亡模型中表达升高,将其过表达能够促进H2O2诱导的HT-29细胞凋亡作用,表明miR-23可能参与了H2O2诱导的细胞凋亡过程。

Objective To observe the expression of microRNA in HEOE-induced apoptosis of HT-29 colon adenocarcinoma cell line and explore the effect of miRNA on H_2O_2-induced apoptosis of HT-29 cells. Methods HT-29 colon adenocarcinoma cell line was treated with 500 nM H_2O_2 for 48 hours to establish apoptotic cell model. Total RNA was isolated with Trizol regent, and the expression of microRNA was determined by Real-time PCR. Combinated plasmid of Psuper was used to over-express miRNA in HT- 29 cells. After transfection for 48 hours, the expression of miRNA was monitored using real-time PCR. Flow cytometry was used to examin the apoptosis of HT-29 cells induced by H_2O_2. Results Compared with control, the expressions of miR-22, miR-24, miR-26, miR-30, miR-181and miR-214 were not altered in H_2O_2-induced HT-29 cells. In contrast, miR-23 was significantly up-regulated in H_2O_2-induced HT-29 cells. Psuper-miR-23 could markedly over-expressed miR-23 in HT-29 cells after transfection for 48 hours. Over-expression of miR-23 for 48 hours could significantly promote apoptosis of HT-29 cells induced by H_2O_2. Conclusion MiR-23 is up-regulated in H_2O_2-induced HT-29 colon adenocarcinoma cell line, and miR-23 could promote apoptotic effects of HT-29 ceils induced by H_2O_2.