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pSi-lencer4.1-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。
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 lucifer- ase 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 ceils effectively. The exogenous miR-147a over-ex- pression 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.
出处
《山东大学学报（医学版）》
CAS
北大核心
2012年第12期25-30,共6页
Journal of Shandong University:Health Sciences
基金
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)
