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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 changes of microRNA (miRNA) expression in HT-29 colon cancer cells induced by hydrogen peroxide (H2O2) and its role in apoptosis. Methods HT-29 colon cancer cells were cultured in vitro and treated with 500 nM H 2 O 2 for 48 h to construct a model of HT-29 cell apoptosis. After routine RNA extraction, reverse transcription reaction was carried out by using miRNA loop-loop primers and real-time fluorescence Quantitative PCR analysis of miRNAs expression levels. The Psuper overexpression plasmid for miR-23 was constructed and transfected into HT-29 cells. Real-time fluorescence quantitative PCR was used to analyze the overexpression efficiency of Psuper-miR-23 in HT-29 cells. MiR-23 was overexpressed in H2O2-induced HT-29 cells for 48 h and the apoptosis of HT-29 cells was detected by flow cytometry. Caspase 3 expression was detected by quantitative PCR. Results The expression levels of miR-22, miR-24, miR-26, miR-30, miR-181 and miR-214 in H2O2-induced HT- The expression difference was not significant, while the expression level of miR-23 in treatment group was significantly increased. Psuper-miR-23 overexpression plasmid transfected HT-29 cells can significantly increase the expression of miR-23. Overexpression of miR-23 in H2O2-induced HT-29 cells can promote HT-29 cell apoptosis. Conclusions miR-23 is up-regulated in H2O2-induced HT-29 cell apoptosis model. Overexpression of miR-23 can promote H2O2-induced HT-29 cell apoptosis, indicating that miR-23 may be involved in H2O2-induced apoptosis process.