HLCDG1基因siRNA表达质粒的构建及其对A549细胞周期和增殖的影响

目的:本研究旨在应用RNA干扰技术，诱导转基因肺癌细胞HLCDG1基因沉默并观察其对肺癌细胞周期和增殖的影响。 方法:构建HLCDG1基因短小干扰双链RNA表达载体，筛选HLCDG1基因表达沉默的肺癌细胞株并应用MTT和流式细胞仪分析这一肺癌细胞株增殖和细胞周期分布状况。 结果:根据siRNA表达载体的设计原则，构建了能在哺乳动物细胞稳定表达的siRNA载体5个（4个位点匹配实验组和1个位点错配对照组），依次命名为pHL-si-1、pHL-si-2、pHL-si-3、pHL-si-4 和 pHL-si-c。上述载体分别与表达HLCDG1的重组体pcDNA3.1(+)/HLCDG1共转染A549细胞，筛选到一株共转染pHL-si-1载体和pcDNA3.1(+)/HLCDG1重组体的A549细胞，它几无HLCDG1基因表达，这株细胞暂命名为A549-HLCDG1-si-1。MTT和流式细胞仪分析表明，A549-HLCDG1-si-1细胞增殖能力明显提高，进入S期和G2+M期增多，滞留G1期减少。 结论:采用RNAi技术特异阻断HLCDG1基因表达，验证了HLCDG1基因对A549肺癌细胞有生长抑制作用。

Construction of HLCDG1 gene siRNA expression vector and its regulation on cell cycle and proliferation in A549 cells

AIM: HLCDG1 is a novel gene cloned recently, and its expression inhibits significantly the growth of A549 cells and tumorigenesis of A549 cells transplanted in nude mice. In this study, our aim was to construct HLCDG1 gene short/small interference double-strand RNA (siRNAs) expression vector and to observe its influence on cell cycle and proliferation of A549 cells. METHODS: Using RNA interference (RNAi) techniques, a DNA vector-driven siRNAs expression vector was constructed, and a lung carcinoma cell line stably expressing siRNAs was also selected. Sequentially, using flow cytometry analysis and MTT assay, the changes of cell cycle and cell proliferation in this cell line were observed. RESULTS: Four site-match and one site-mismatch plasmids were constructed, which were named pHL-si-1, pHL-si-2, pHL-si-3, pHL-si-4 and pHL-si-c. These plasmids were co-transfected with a pcDNA3.1(+)/HLCDG1 plasmid into A549 cells, respectively. Among five co-transfected A549 cell lines, a A549 cell line co-transfected by the pcDNA3.1(+)/HLCDG1 and pHL-si-1 plasmids, namely A549-HLCDG1-si-1, showed nearly complete inhibition of HLCDG1 expression. MTT assay and flow cytometry analysis indicated that A549-HLCDG1-si-1 cells, namely the HLCDG1 gene-silencing cells, got a faster growth compared with other HLCDG1 expression cell lines, and that HLCDG1 gene-silencing induced A549-HLCDG1-si-1 cells into S phase and G_2+M phase significantly. CONCLUSION: These results suggest that the HLCDG1 gene is proved to have a markedly inhibitory effect on growth in A549 lung carcinoma cells. This study might provide some understanding of the biological function and molecular mechanism of HLCDG1 gene.