论文部分内容阅读
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 eukaryotic expression vector of human miR-147a to detect the expression of miR-147a in lung adenocarcinoma A549 cells and its effect on cell proliferation and invasion. Methods The pri-mir-147a gene was amplified by RT-PCR using total RNA of A549 cells as a template and inserted into the expression vector pSi-lencer4.1-CMV neo to construct pSilencer4.1-147a recombinant vector. The expression of miR-147a was detected by qRT-PCR and reporter gene analysis. The effect of exogenous miR-147a overexpression on the proliferation of A549 cells was detected by MTT assay. The exogenous miR-147a overexpression was detected by Matrigel invasion assay On the invasion ability of A549 cells; reporter gene analysis to detect the impact of miR-147a on common tumor signal pathways. Results Restriction endonuclease digestion and DNA sequencing confirmed that pSilencer4.1-147a was constructed correctly. The expression of exogenous miR-147a was detected by qRT-PCR and reporter gene analysis after A549 cells were transfected with pSilencer4.1-147a. MTT analysis and Matrigel invasion assay showed that over-expression of miR-147a significantly inhibited the proliferation and invasion of A549 cells. The reporter gene analysis showed that miR-147a overexpression significantly down-regulated the expression of pGL4.23-AP1, pGL4.23-ELK1 and pGL4. 23-cMYC relative luciferase activity. Conclusions The successfully constructed human miR-147a eukaryotic expression vector can effectively express in lung adenocarcinoma A549 cells and inhibit the proliferation and invasion of A549 cells, which may be related to the effect of miR-147a on EGFR-RAS-MAPK pathway Suppression related.