宫颈癌细胞通过下调miR-301b表达促进基因组DNA损伤修复

Promotion of repair of genomic DNA damage by down-regulating the expression of miR-301b in cervical cancer cells

目的 通过研究miR-301b、C末端结合蛋白作用蛋白(Ctip)基因表达在DNA损伤修复过程中的作用以及miR-301b影响Ctip基因表达的作用机制,揭示宫颈癌细胞修复DNA损伤的新机制。方法 (1)Siha细胞经肿瘤坏死因子-α(TNF-α)处理、miR-301b模拟物、抑制物转染以及pcDNA3.1/Ctip与miR-301b模拟物共转染后,利用流式细胞术检测平台和彗星实验测定细胞内DNA损伤标志物γ-H2AX蛋白平均表达水平和DNA彗星状尾距;(2)采用实时定量聚合酶链式反应(PCR)检测DNA损伤修复过程中细胞内miR-301b、Ctip mRNA的水平以及转染miR-301b模拟物、抑制物后细胞内Ctip mRNA的水平;(3)采用Western-blot蛋白印迹试验检测转染miR-301b模拟物、抑制物后细胞内Ctip蛋白的水平;(4)应用生物信息学预测miR-301b在Ctip mRNA上的靶定位点;(5)应用荧光报告实验检测转染miR-301b模拟物、抑制物后细胞内mRNA上存在miR-301b靶定位点的报告基因的表达水平。结果 (1)与对照相比,刺激1.5和3 h后,肿瘤坏死因子-α(TNF-α)使细胞内γ-H2AX蛋白平均水平和DNA平均尾距升高,刺激6 h后,TNF-α对胞内γ-H2AX蛋白平均水平以及DNA平均尾距无影响;转染miR-301b模拟物升高γ-H2AX蛋白平均水平和DNA尾距,转染miR-301b抑制物降低γ-H2AX蛋白平均水平和DNA平均尾距;与miR-301b模拟物共转染的pcDNA3.1/Ctip降低细胞内γ-H2AX蛋白平均水平和DNA平均尾距;(2)TNF-α刺激使细胞内miR-301b水平降低,Ctip mRNA水平升高;转染miR-301b模拟物降低Ctip mRNA水平,转染miR-301b抑制物升高Ctip mRNA水平;(3)转染miR-301b模拟物降低Ctip蛋白水平,转染miR-301b抑制物升高Ctip蛋白水平;(4)miR-301b靶定Ctip mRNA的3208至3214核苷酸位点;(5)mRNA上存在miR-301b靶定位点的报告基因的表达水平在miR-301b模拟物转染后降低,在miR-301b抑制物转染后升高。结论 Siha细胞通过下调miR-301b表达增强Ctip基因表达促进基因组DNA的损伤修复。

Objective To uncover a novel pathway which was involved in the DNA damage repair on the basis of studying the roles of the expressions of miR-301b and C-terminal binding protein interacting protein (Ctip) genes in the DNA damage repairs and the impacts of miR-301bs on the Ctip gene expressions and the mechanism underlying the actions of miR-301bs. Methods (1)After Siha cells treated with tumor necrosis factor-α(TNF-α),transfected with miR-301b mimics or inhibitors and cotransfected with pcDNA3.1/Ctip and miR-301b mimics,the average expression levels of γ-H2AX proteins and oliver tail moments of DNA indicating the damage in DNA in the cells were measured on the flowcytometry system and in comet assays.(2)The levels of miR-301bs and Ctip mRNAs in the cells which underwent DNA damage repairs and that of Ctip mRNAs in the cells transfected with miR-301b mimics or inhibitors were determined in quantitative real-time PCR assays.(3)The levels of Ctip proteins in the cells transfected with miR-301b mimics or inhibitors were detected in Westernblot assays.(4)Bioinformatic tools were utilized to find out the potential targeted sites of miR-301b on the Ctip mRNA.(5) The expression levels of the report genes whose mRNAs contain the miR-301b targeted sites in cells were measured in fluorescence report assays. Results (1)When compared with controls,TNF-α made the levels of γ-H2AX proteins and DNA oliver tail moments increased in the cells which were incubated with TNF-α for 1.5 and 3 hours but no changes in the levels of γ-H2AX proteins and oliver tail moments were found in the cells which incubated with TNF-αfor 6 hours.The transfections of miR-301b mimics increased the levels of γ-H2AX proteins and DNA oliver tail moments while the transfections of miR-301b inhibitors decreased the levels and oliver tail moments.(2)TNF-α administrations reduced the miR-301b levels but elevated the Ctip mRNA levels in the cells,and the transfections of miR-301b mimics resulted in the reductions in the levels of Ctip mRNAs while the transfections of inhibitors led to the increases in the levels.(3)The transfections of miR-301b mimics induced the decreases in the levels of Ctip proteins while the transfections of inhibitors caused the augments in the levels.(4)MiR-301b targeted the sequence located at positions 3208-3214 of Ctip mRNA.(5)The expression levels of report genes whose mRNAs containing miR-301b targeted sites were downregulated following the transfections of miR-301b mimics but upregulated following the transfections of miR-301b inhibitors. Conclusion Downregulation of miR-301b which causes the enhanced gene expression of Ctip contributes to the DNA damage repair in cervical cancer cells.