目的 观察miR-130b对宫颈癌细胞修复基因组双链DNA断裂作用的影响, 并探讨其机制.方法 将宫颈癌细胞Siha分成6组, 分别转染细胞周期蛋白依赖性蛋白激酶抑制物1A (CDKN1A) 基因过表达质粒pc DNA3. 1-CDKN1A (CDKN1A组) 、pc DNA3. 1空载质粒 (pc DNA3. 1组) 、miR-130b mimics (miR-130b组) 、miR-130b阴性对照核酸 (NC组) , 共转染pc DNA3. 1-CDKN1A和miR-130b mimics (CDKN1A+miR-130b组) 、pc DNA3. 1和miR-130b mimics (pc DNA3. 1+miR-130b组) .TNF-α刺激细胞, 用彗星试验测定基因组DNA尾距, 流式细胞术测定磷酸化组蛋白2A变异体 (γ-H2AX) 蛋白; Real-time PCR、Western blotting法检测CDKN1A mRNA及其蛋白.分别将pc DNA3. 1/EGFP、pc DNA3. 1/EGFP-CDKN1A-wtUTR (野生型) 、pc DNA3. 1/EGFP-CDKN1A-mutUTR (突变型) 与miR-130b mimics或NC共转染Siha细胞, 测定报告基因荧光相对活性;以TNF-α和DNA损伤增强剂AZD2461刺激各组细胞, 用流式细胞术测定细胞凋亡水平.结果 与pc DNA3. 1组比较, CDKN1A组细胞DNA尾距、γ-H2AX蛋白表达水平降低 (P均<0. 05) ;与NC组比较, miR-130b组细胞DNA尾距、γ-H2AX蛋白表达水平、细胞凋亡率升高, CDKN1A mRNA、蛋白表达水平下降 (P均<0. 05) ;与pc DNA3. 1+miR-130b组比较, CDKN1A+miR-130b组细胞DNA尾距、γ-H2AX蛋白表达水平、细胞凋亡率下降 (P均<0. 05) ;与共转染NC细胞相比, 共转染miR-130b mimics使野生型细胞荧光相对活性降低 (P <0. 05) , 而未改变突变型细胞荧光相对活性.结论 miR-130b通过直接靶定CDKN1A mRNA抑制其表达干扰宫颈癌细胞修复双链DNA断裂位点, 从而促进细胞凋亡.%Objective To observe the impact of miR-130 b on the repair of DNA double strand breaks in cervical cancer cells and to discuss the mechanism underlying them. Methods Siha cells were classified into 6 groups. The cells transfected with pc DNA 3. 1-CDKN1A ( cyclin-dependent kinase inhibitor 1A) , pc DNA 3. 1, miR-130 b mimics and the negative control of miR-130 b were taken as the CDKN1 A, pc DNA 3. 1, miR-130 b and NC groups in turn. The ones cotransfected with miR-130 b and pc DNA 3. 1-CDKN1 A and those with miR-130 b and pc DNA 3. 1 were taken as the CDKN1 A + miR-130 b and pc DNA 3. 1 + miR-130 b groups. Cells were stimulated with TNF-α after the transfection. DNA oliver tail moments ( OTM) were determined in comet assays and the phosphorylated H2 AX histone protein variant ( γ-H2AX) levels were measured by flow cytometry. The CDKN1 A mRNA and its protein levels were measured by using semiquantitative real-time PCR and Western blotting. pc DNA 3. 1/EGFP, pc DNA 3. 1/EGFP-CDKN1A-wtUTR ( wild type) and pc DNA 3. 1/EGFP-CDKN1A-mutUTR ( mutant type) were transfected into Siha cells with miR-130 b mimics or NC, and the expression levels of reporter genes were determined. We used TNF-α and DNA damage enhancer AZD2461 to stimulate the cells in each group, and then used flow cytometry to determine the level of apoptosis. Results Compared with the pc DNA 3. 1 group, both the oliver tail moment and the γ-H2 AX protein level reduced in the cells of pc DNA 3. 1-CDKN1 A group ( both P < 0. 05) ; compared with the NC group, the oliver tail moment and the γ-H2 AX protein level increased whereas the CDKN1 A mRNA and protein levels decreased in the cells of miR-130 b group ( all P < 0. 05) . Compared with the pc DNA 3. 1 + miR-130 b group, the oliver tail moment, γ-H2 AX protein level and apoptosis rate decreased ( all P < 0. 05) ; compared with NC cotransfection, the cotransfection with miR-130 b mimics reduced the fluorescence levels within cells, but did not change the fluorescent relative activity of the mutant cells. Conclusion The miR-130 b hampers the repair of DNA double strand breaks in cervical cancer cells by inhibiting CDKN1 A gene expression through directly targeting CDKN1 A mRNA to increase the apoptosis rates.
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