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山东大学学报(医学版) ›› 2012, Vol. 50 ›› Issue (12): 25-30.

• 基础医学 • 上一篇    下一篇

人miR-147a真核表达载体的构建及其对肺癌细胞增殖和侵袭的影响

于洋1,赵健2,钟铠泽2,刘春艳1,李艺3,史婧3,陈蔚文1,姜安丽1   

  1. 1. 山东大学医学院生物化学与分子生物学研究所, 济南 250012;
    2. 山东大学齐鲁医院胸外科,  济南 250012;3. 山东大学医学院, 济南 250012
  • 收稿日期:2012-07-06 出版日期:2012-12-10 发布日期:2012-12-10
  • 通讯作者: 陈蔚文(1971- ),女,副教授,硕士生导师,主要从事肿瘤分子生物学研究。E-mail: chenweiwen@sdu.edu.cn
  • 作者简介:于洋(1987- ),男,硕士研究生,主要从事肿瘤分子生物学研究。E-mail: kingyuyang0325@163.com
  • 基金资助:

    山东省自然科学基金项目(ZR2009CM044);山东省优秀中青年科学家科研奖励基金计划(2006BS03066,BS2009SW042)

Construction of eukaryotic expression vector for human miR-147a and
its effects on the proliferation and invasion of lung cancer cells

YU Yang1,  ZHAO Jian2, ZHONG Kai-ze2,  LIU Chun-yan1, LI Yi3,
SHI Jing3, CHEN Wei-wen1,  JIANG An-li1   

  1. 1. Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, China;
    2. Department of Thoracic Surgery, Qilu Hospital of Shandong University, Jinan 250012, China;
    3.  School of Medicine, Shandong University, Jinan 250012, China
  • Received:2012-07-06 Online:2012-12-10 Published:2012-12-10

摘要:

目的   构建人miR-147a真核表达载体,检测其在肺腺癌细胞A549中的表达及对细胞增殖和侵袭能力的影响。方法   以A549细胞总RNA为模板,RT-PCR扩增miR-147a的前体序列(pri-mir-147a),插入表达载体pSilencer4.1-CMV neo,构建pSilencer4.1-147a重组载体,转染A549细胞,qRT-PCR和报告基因分析检测miR-147a的表达;MTT分析检测外源性miR147a过表达对A549细胞增殖能力的影响;Matrigel侵袭实验检测外源性miR-147a过表达对A549细胞侵袭能力的影响;报告基因分析检测miR147a对常见肿瘤信号途径的影响。结果   限制性酶切和DNA测序证实pSilencer4.1-147a构建正确;pSilencer4.1-147a转染A549细胞后,qRT-PCR和报告基因分析检测到明显的外源性miR-147a表达;MTT分析和Matrigel侵袭实验结果显示,miR-147a过表达可明显抑制A549细胞的增殖和侵袭能力;报告基因分析结果显示,miR-147a过表达可显著下调pGL4.23-AP1、pGL4.23-ELK1、pGL4.23-cMYC的相对荧光素酶活性。结论   成功构建的人miR-147a真核表达载体能够在肺腺癌细胞A549中有效表达,并对A549细胞的增殖和侵袭产生抑制作用,该作用可能与miR-147a对EGFR-RAS-MAPK途径的抑制有关。

关键词: microRNA;miR-147a;肺肿瘤;增殖;侵袭

Abstract:

Objective   To construct the recombinant eukaryotic expression vector for human miR-147a and determine its effects on the proliferation and invasion of lung adenocarcinoma cell A549 due to miR-147a expression. Methods   pri-mir-147a sequence was amplified by RT-PCR using total RNA from A549 cells and was inserted into the pSilencer4.1CMV neo expression vector to construct the recombinant pSilencer4.1-147a vector, which was transfected into A549 cells, followed by qRT-PCR and reporter gene assay to detect the expression of miR-147a. MTT assay was utilized to determine the effect of exogenous miR-147a over-expression on the proliferation of A549 cells. Matrigel invasion assay was performed to detect the effect of exogenous miR-147a over-expression on the invasion of A549 cells. Finally,reporter gene assay was used to observe the effects of miR-147a on common tumor signal pathways. Results   The construction  of pSilencer4.1-147a was confirmed to be correct by restriction enzyme digestion and DNA sequencing. qRT-PCR and reporter gene assay displayed significant ectopic expression of miR-147a after the transfection of pSilencer4.1-147a in A549 cells. MTT assay and Matrigel invasion assay showed that miR-147a significantly inhibited the proliferation and invasion ability of A549 cells. Reporter gene assay showed that miR-147a significantly down-regulated relative luciferase activity of pGL4.23-AP1, pGL4.23-ELK1 and pGL4.23-cMYC. Conclusion   The eukaryotic expression vector of human miR-147a is constructed successfully and expressed in A549 cells effectively. The exogenous miR-147a over-expression shows inhibitive effects on the proliferation and invasion of A549 cells, which maybe relevant to the inhibition of EGFR-RAS-MAPK pathway by miR-147a.

Key words: microRNA; miR-147a; Lung neoplasms; Proliferation; Invasion

中图分类号: 

  • R734
[1] . 吉非替尼联合塞来昔布对肺腺癌细胞凋亡和EGFR、COX2表达的影响[J]. 山东大学学报(医学版), 2009, 47(8): 37-41.
[2] 郝可可1,朱际平2,于力克1. Survivin检测在肺肿瘤胸腔积液诊断中的临床价值[J]. 山东大学学报(医学版), 2010, 48(12): 129-133.
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