Effects of Dnmt1 gene on proliferation and apoptosis of gastric cancer cell line AGS

    CHEN Daoª²Rong, WANG Piª²Long, YANG Xueª²Qin, HUANG Aiª²Long, ZHANG Bingª²Qiang, TAO Xiaoª²Hong

    Department of Gastroenterology, First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China, Institute of Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China, Department of Gastroenterology, Chongqing People¬ðs Hospital of Banan District, Chongqing 401320, China

    ¡¾Abstract¡¿ AIM: To design a recombinant plasmid pshRNAª²dnmt1 by RNA interference and to assess its effects on DNA methyltransferase (Dnmt1) gene expression in gastric cancer cell line AGS, and on the proliferation and apoptosis of AGS cells in vitro. METHODS: The eukaryotic expression plasmid pshRNAª²Dnmt1, containing the sequence of shRNA, was cloned and transfected into AGS cells. The mRNA levels of gene Dnmt1 were determined by RTª²PCR, the protein expression levels by Western Blot, the cell survival index by MTT assay, and the apoptosis was also evaluated by AO/EB, electron microscope and TUNEL. RESULTS: A recombinant plasmid pshRNAª²Dnmt1 was successfully constructed and confirmed by sequencing analysis. PshRNAª²Dnmt1 induced a 21.63% inhibition of Dnmt1 mRNA after 24 h treatment, 52.97% after 48 h, and 72.06% after 72 h. Dnmt1 protein levels in AGS cells appeared to reduce, with inhibition rate of 28.24% after 24 h of treatment, 68.54% after 48 h, and 81.47% after 72 h. MTT results showed that the cell survival rates of AGS cells after transfected with pshRNAª²Dnmt1 at 24 h, 48 h and 72 h respectively were 79.49%, 51.63% and 39.16%. AO/EB, electron microscope and TUNEL test indicated: after transfection with the recombinant plasmid, there existed a lot of apoptotic and necrotic cells, and the apoptosis rate increased from about 5% to 35%. CONCLUSION: The recombinant plasmid pshRNAª²Dnmt1 can efficiently and specifically inhibit the expression of gene Dnmt1, inhibit the proliferation of AGS cells, and induce the apoptosis. ......