MicroRNA-34a调控细胞衰老的研究

Research on Regulation of Cell Senescence by MiRNA-34a

目的探讨微RNA-34a(miRNA-34a)对细胞通过调控沉默交配型信息调节因子2同源蛋白1(SIRT1)的表达,从而引起细胞衰老的作用。方法构建pre-miRNA-34a表达载体,并将其和一个不针对任何基因的微小RNA-1792(miRNA-1792)表达载体(其他实验室赠送)分别转染至HEK293和HUVEC细胞内,用RT-PCR、Western blot检测各细胞组SIRT1mRNA与蛋白表达水平,以未转染的细胞为对照;将HUVEC细胞分为转染不同质粒、未转染质粒和用SIRT1激活剂白藜芦醇(终浓度1μmol/L,作用2h)处理HUVEC细胞(HUVEC-Res)组,中性彗星电泳分析H2O2作用2h后4组HUVEC细胞的双链损伤情况;用阿霉素处理上述4组HUVEC细胞2h,β-半乳糖甘酶(SA-β-gal)染色检测各细胞不同时间点的衰老情况。结果测序结果表明pre-miRNA-34a表达载体构建成功;RT-PCR、Western blot显示HEK293-pre-miRNA-34a和HUVEC-pre-miRNA-34a中SIRT1mRNA和蛋白质表达的水平明显下降(P<0.001);中性彗星电泳检测发现经H2O2处理后,HUVEC-Res组DNA损伤最轻,而HUVEC-miRNA-34a组DNA损伤程度最为严重;SA-β-gal染色显示DNA损伤后HUVEC-miRNA-34a衰老程度较其它3组严重,而HUVEC-Res组的衰老程度最为轻微。结论 miRNA-34a通过抑制SIRT1表达调控细胞衰老。

Objective To determine miRNA-34a regulated cell senescence indirectly through targeting silent mating-type information regulation 2 homologue 1 （SIRT1） in vitro experiment. Methods A constructed pre- miRNA -34a expression vector and a miRNA-1792 expression vector （not directly against any gene） were transfected into HEK293 and HUVEC cell lines respectively. The expression levels of SIRT1 in each cell groups were detected by RT-PCR and Western blot. The HUVEC cells were divided into different group： transfected with pre-miRNA- 34a expression vector （HUVEC-pre-miRNA-34a）, transfected with miRNA-1792 expression vector （HUVEC-pre- miRNA-1792）, treated HUVEC cell with SIRT1 activator resveratrol （final concentration 1μmol/L, treatment for 2 h）（HUVEC-Res）, and HUVEC cells without any treatment as the control. Comet assay was applied to detect the oxidative damage of above-mentioned cells after H2 02 treatment for 2 h, and beta-galactosidase （SA-[3-gal） staining was used to detect the senescence of them in different time points after doxorubicin treatment for 2 h. Results Pre-miRNA-34a expression vector was constructed successfully by sequencing confirmation. RT-PCR and Western blot indicated that the overexpression of miRNA-34a down regulated mRNA and protein level of SIRT1 in HEK293- miRNA-34a and HUVEC-miRNA-34a cell groups （P〈0. 001）. Comet assay revealed that HUVEC-miRNA-34a cell group was the most sensitive to H2O2 treatment, and the DNA damage of HUVEC-Res cell group was the most minor. HUVEC-miRNA-34a cell group displayed higher frequency of SA-β-gal staining than that of other cell groups. Conclusion miRNA-34a regulated cell senescence indirectly through targeting SIRT1.