miR-146a前体多态性对宫颈癌细胞增殖的影响
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

江苏省科技厅基础研究计划(自然科学基金)(BK2012878)


Polymorphism effect of Pre-miR-146a on the proliferation of cervical cancer cells
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    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-

    Abstract:

    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.

    参考文献
    相似文献
    引证文献
引用本文

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(自然科学版),2016,(6):676-681

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2015-09-22
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2016-06-15
  • 出版日期:
通知关闭
郑重声明