MiR-23a质粒的构建及其在胃腺癌细胞系MGC803中的表达

Construction of the vector of miR-23a and expression in gastric adenocarcinoma cell line MGC803

目的构建表达微小RNA-23a(miR-23a)的真核表达质粒,为进一步研究小RNA分子miR-23a在胃腺癌细胞系MGC803中的功能奠定良好的实验基础。方法首先设计并合成扩增miR-23a前体基因全长的PCR引物,提取胃腺癌细胞系MGC803基因组为模板,PCR扩增大小为324 bp的目的基因;PCR产物经过EcoR I和Bgl II酶切后,与线性化的pcDNA3载体连接后转化大肠杆菌,扩增、纯化获得所需质粒,以琼脂糖凝胶电泳、酶切鉴定以及基因测序鉴定其分子质量和插入片段的序列;构建的质粒经脂质体方法转染MGC803细胞,实时定量PCR检测转染细胞MGC803中miR-23a的表达水平,经MTT实验、平板克隆形成实验、软琼脂克隆形成实验、TUNEL实验及Transwell实验,观察过表达miR-23a后对MGC803细胞增殖、凋亡及侵袭能力的影响。结果纯化质粒的相对分子质量为5.7 kb,酶切及测序鉴定结果符合目的条带大小(324 bp),插入的寡核苷酸序列与miR-23a前体基因序列完全相符;实时定量PCR结果表明pcDNA3/miR-23a能够有效表达miR-23a;MTT、平板克隆形成及软琼脂克隆形成实验结果显示,过表达miR-23a可以促进MGC803细胞活性;TUNEL实验结果显示,过表达miR-23a可以减少胃腺癌细胞MGC803凋亡的发生;Transwell实验结果显示miR-23a可以促进胃腺癌细胞MGC803的侵袭能力。结论构建的真核表达质粒在胃腺癌细胞MGC803中有效表达miR-23a,能够促进细胞活性及侵袭能力,并能抑制凋亡的发生。

[Objectives] To construct an expression vector of miR-23a and investigate the expression of this gene in gastric adenocarcinoma cell line MGC803. [Methods] The miR-23a gene was cloned from the genome of gastric adenocarcinoma cell line MGCS03 and the product of PCR was digested by EcoR I and Bgl ]I. Then the miR-23a fragment was inserted into the pcDNA3 plasmid to get the new plasmid pcDNA3/ miR-23a. The new plasmid was identified by digestion of restriction enzyme and sequencing. Subsequently,pcDNA3/miR-23a plasmid was transfected into the gastric adenocarcinoma cell line MGC803 by lipofection. The mRNA level of miR-23a was detected with quantitative real-time PCR. Then the changes of cell growth were detected with MTI？ assay and colony formation assay. And the TUNEL assay or the transwell assay was adopted to detect the effects on the apoptosis or the invasion of MGCS03. [Results] The constructed plasmid was about 5.7 kb long, and the inserted sequence was identical with design, with no aberrations such as mu- tation, deletion, or insertion. The gastric adenocarcinoma cell line MGC803 can express miR-23a after trans- fection with pcDNA3/miR-23a plasmid. After over-expression of miR-23a, the MGC803 cell growth activity, the colony formation activity and non-anchoring growth activity were all promoted. And the apoptosis of MGC803 was attenuated but the invasion was enhanced. [Conclusion] The plasmid pcDNA3/miR-23a can be successfully constructed and can express in gastric adenocarcinoma cell line MGC803 after transfection. MiR- 23a can promote the cell growth activity and invasion but inhibit the apoptosis of MGC803.