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.