文章摘要
胡启明,丁 波,崔毓桂,周德兰,梁佳乐,韩男男,韩素萍.miR-146a前体多态性对宫颈癌细胞增殖的影响[J].南京医科大学学报,2016,(6):676~681
miR-146a前体多态性对宫颈癌细胞增殖的影响
Polymorphism effect of Pre-miR-146a on the proliferation of cervical cancer cells
投稿时间:2015-09-22  
DOI:10.7655/NYDXBNS20160607
中文关键词: 宫颈癌  真核表达载体  miR-146a  细胞增殖  肿瘤坏死因子受体相关激酶-6
英文关键词: cervical cancer  eukaryotic expression plasmid  miR-146a  cell proliferation  TNF receptor-associated factor 6
基金项目:江苏省科技厅基础研究计划(自然科学基金)(BK2012878)
作者单位
胡启明 南京医科大学第一附属医院妇产科,江苏 南京 210036 
丁 波 东南大学附属中大医院妇产科,江苏 南京 210009 
崔毓桂 南京医科大学第一附属医院妇产科,江苏 南京 210036 
周德兰 南京医科大学第一附属医院妇产科,江苏 南京 210036 
梁佳乐 南京医科大学第一附属医院妇产科,江苏 南京 210037 
韩男男 南京医科大学第一附属医院妇产科,江苏 南京 210038 
韩素萍 南京医科大学第一附属医院妇产科,江苏 南京 210039 
摘要点击次数: 530
全文下载次数: 349
中文摘要:
      目的:通过转染miR-146a前体(Pre-miR-146a)多态性真核表达质粒,观察miR-146a多态性对miR-146a表达情况和宫颈癌HeLa细胞增殖能力的影响并探讨可能的作用机制?方法:PCR扩增含多态性位点的Pre-miR-146a目的片段,连接入pcDNA3.1(+)质粒表达载体得到Pre-miR-146a-G及Pre-miR-146a-C重组质粒,将重组质粒转染宫颈癌HeLa细胞?实时荧光定量PCR(real-time PCR)检测miR-146a的表达,CCK8法检测HeLa细胞的增殖情况,real-time PCR以及Western blot检测miR-146a靶基因肿瘤坏死因子受体相关激酶-6(TNF receptor-associated factor 6,TRAF6)的表达?结果:成功将miR-146a多态性前体片段克隆入pcDNA3.1(+)真核表达质粒,获得Pre-miR-146a-G及Pre-miR-146a-C重组质粒;转染重组质粒能够在HeLa细胞内高效和特异地表达miR-146a(P < 0.05),且转染Pre-miR-146a-C质粒组miR-146a表达量较Pre-miR-146a-G质粒组高,差异有统计学意义(P < 0.05);HeLa细胞转染重组质粒48 h后,其细胞存活率较对照组显著增加(P < 0.05),细胞内TRAF6的蛋白水平显著下降(P < 0.05),但TRAF6的mRNA水平无变化,差异无统计学意义(P > 0.05)?结论:Pre-miR-146a多态性位点能影响成熟miR-146a表达,过表达miR-146a可能通过下调TRAF6基因抑制NF-κB信号通路,从而促进HeLa细胞增殖?
英文摘要:
      Objective:To transfect the eukaryotic expression plasmid of Pre-miR-146a with a single nucleotide polymorphism (SNP) and observe the expression efficiency of miR-146a in cervical cancer HeLa cells,and then explore the possible mechanism of miRNA-146a on the proliferation of HeLa cells. Methods:We directly cloned a 370-bp fragment including miR-146a precursor from the human gDNA into the pcDNA3.1 (+) plasmid to construct recombinant Pre-miR-146a plasmid by PCR. A Pre-miR-146a-G or Pre-miR-146a-C was transfected into HeLa cell line and the expression of miR-146a was analyzed by real-time PCR. Cell proliferation was measured by cell counting kit-8 assay. Real-time PCR and Western blotting assays were performed to assess the expression of TNF receptor-associated factor 6 (TRAF6). Results:The fragment including miR-146a precursor was successfully cloned into the pcDNA3.1 (+) plasmid and the recombinant expression plasmid Pre-miR-146a-G or Pre-miR-146a-C was constructed. After transfection of Pre-miR-146a-G or Pre-miR-146a-C recombinant plasmid into HeLa cell line,the expression of miR-146a was significantly increased (P < 0.05). The miR-146a expression of the Pre-miR-146a-C plasmid transfected group was higher than that of the Pre-miR-146a-G plasmid group (P < 0.05). After transfection with recombinant plasmid 48 h,the cell viability of HeLa cell line was increased (P < 0.05) and TRAF6 protein expression was down-regulated (P < 0.05),but the mRNA level of TRAF6 did not changed (P > 0.05). Conclusion:The SNP in Pre-miR-146a enhanced the expression of miR-146a and promoted the proliferation of HeLa cells,probably by inhibiting the NF-κB signaling pathway by down-regulating TRAF6.
查看全文   查看/发表评论  下载PDF阅读器
关闭