人lncRNA-GACAT2过表达载体的构建及其对肺癌细胞增殖和干性的影响

Construction of human lncRNA-GACAT2 over-expression vector and its effect on proliferation and stemness of lung cancer cells

目的:探讨长链非编码RNA(lncRNA)胃癌相关转录本2(GACAT2)基因对肺癌A549和H1299细胞增殖和干性的影响,阐明lncRNA GACAT2基因在肺癌发生发展中的作用。方法:实时荧光定量PCR(RT-qPCR)法扩增lncRNAGACAT2基因的全长序列,克隆至pc3.1(+)真核表达载体,采用菌液PCR法和基因测序法检测GACAT2过表达载体的构建情况。将pc3.1-GACAT2过表达质粒和对照质粒pc3.1分别转入人肺癌A549和H1299细胞,采用G418筛选并建立稳定过表达GACAT2的A549和H1299细胞,分为空载体组(转染pc3.1空载体)和pc3.1-GACAT2组(转染pc3.1-GACAT2重组质粒)。采用RT-qPCR法检测2组A549和H1299细胞中GACAT2 mRNA表达水平,CCK-8法检测2组细胞增殖活性,细胞克隆形成实验检测各组细胞克隆形成数,干细胞成球实验观察2组细胞成球数。结果:成功构建GACAT2真核过表达载体。与空载体组比较,pc3.1-GACAT2组A549和H1299细胞中GACAT2 mRNA表达水平升高(P<0.05或P<0.01),细胞增殖活性降低(P<0.05或P<0.01),细胞克隆形成数明显降低(P<0.05或P<0.01),细胞成球数明显降低(P<0.05或P<0.01)。结论:过表达人lncRNA GACAT2基因能抑制肺癌A549和H1299细胞的增殖能力和细胞干性。

Objective:To discuss the effect of long non-coding RNA(lncRNA)gastric cancer related transcript 2(GACAT2)gene on the proliferation and stemness of the lung cancer A549 and H1299 cells,and to clarify the effect of lncRNA GACAT2 gene on the occurrence and development of lung cancer.Methods:The full-length sequence of lncRNA GACAT2 gene was amplified by real-time fluorescence quantitative PCR(RT-qPCR)method,and was cloned into the pc3.1(+)eukaryotic expression vector.The construction of GACAT2 over-expression vector was confirmed by bacterial liquid PCR method and gene sequencing.The pc3.1-GACAT2 over-expression plasmid and control plasmid pc3.1 were separately transfected into the human lung cancer A549 and H1299 cells.The A549 and H1299 cells with stable overexpression of GACAT2 were established by G418 screening,and the cells were divided into blank vector group(transfected with pc3.1 empty vector)and pc3.1-GACAT2 group(transfected with pc3.1-GACAT2 recombinant plasmid).RT-qPCR method was used to detect the expression levels of GACAT2 mRNA in the A549 and H1299 cells in two groups;CCK-8 assay was used to detect the proliferation activities of the cells in two groups;cell clone formation assay was used to detect the clone formation numbers of the cells in two groups;stem cell sphere formation experiment was used to detect the sphere formation numbers of the A549 and H1299 cells in two groups.Results:The eukaryotic over-expression vector of GACAT2 was successfully constructed.Compared with blank vector group,the expression levels of GACAT2 mRNA in the A549 and H1299 cells in pc3.1-GACAT2 group were increased(P<0.05 or P<0.01),the cell proliferation activities were decreased(P<0.05 or P<0.01),the clone formation numbers were decreased(P<0.05 or P<0.01),and the sphere formation numbers were decreased(P<0.05 or P<0.01).Conclusion:Over-expression of human lncRNA GACAT2 gene can inhibit the proliferation and stemness of the lung cancer A549 and H1299 cells.