[Effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer: experiments in vitro and in vivo].
OBJECTIVE
To investigate the effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer.


METHODS
Recombinant eukaryotic expression plasmid pshRNA-DNMT1 containing the sequence of the gene of DMNT1 that methylates the specific pyrimidine residue in the DNA promoter region was constructed. Human gastric cells of the line AGS were cultured and transfected with pshRNA-DNMT1. Western blotting was used to detect the protein expression of DNMT1 of the AGS cells, and RT-PCR was used to detect the mRNA expression of DNMT1 of the AGS cells. MTT method was used to dynamically monitor the surviving cells and the cell apoptotic was observed by electron microscopy and TUNEL method. Forty nude the mice were inoculated with suspension of AGS cells. When the tumor reached the size of 5 - 6 mm in diameter the mice were randomly divided into 5 equal groups to be injected intravenously with PBS, liposome, pTZU6 + 1, pshRNA-DNMT1 of medium dose, and pshRNA-DNBMT1 of large dose for 4 times with an interval of 3 days. The tumor size was measured every day. Three days after the last injection the mice were killed and the tumors were taken out to undergo light and electron microscopy and TUNEL method to detect the cell apoptosis. Immunohistochemistry was used to detect the proliferating cell nuclear antigen (PCNA) of the cells.


RESULTS
The protein and mRNA expression levels of DNMT1 in the cultured AGS cells 24, 48, and 72 hours after transfection of the pshRNA-DNMT1 group were all lower than those of the control group. The numbers of surviving AGS cells of the pshRNA-DNMT1 group became significantly gradually lower than those of the liposome and pTZU6 + 1 groups since 24 hours after transfection (all P < 0.05). The apoptotic rate of AGS cells in the pshRNA-DNMT1 group was 34.78% +/- 0.52%, significantly higher than those of the liposome and pTZU6 + 1 groups (4.86% +/- 0.17% and 5.12% +/- 0.76% respectively, both P < 0.05). The subcutaneous tumors of the mice of the PGS, liposome, and pTZU6 + 1 groups augmented along with time without significant differences among these 3 groups (all P > 0.05). The tumor of the 2 pshDNMT1 groups began to augment since the 5(th) day and began to be reduced in size since the 10(th) day in comparison with the other 3 groups (all P < 0.05), and the tumor size of the pshRNA-DNMT1 (large dose) group was significantly smaller than that of the pshRNA-DNMT1 (medium dose) group 15 days after the injection (P < 0.05). The rates of cell apoptosis of the pshRNA-DNMT1 (large dose) and pshRNA-DNMT1 (medium dose) groups were both significantly higher than those of the other 3 groups (all P < 0.05) and with a sufficient difference between these 2 pshRNA-DNMT1 groups (P < 0.05). PCNA analysis showed that the proliferation activity of the cells in the pshRNA-DNMT1 groups was significantly suppressed.


CONCLUSIONS
The recombinant plasmid pshRNA-NMT1 effectively and specifically inhibits the expression of the gene DNMT1, thus inhibiting the proliferation and inducing the apoptosis of gastric cancer cells.
Chen
Dao-rong
DR
Department of Gastroenterology, First Affiliated Hospital of Chongqing University of Medical Sciences, Chongqing 400016, China. cdrcdrcn@yahoo.com.cn
Wang
Pi-long
PL
Huang
Ai-long
AL
Zhang
Bing-qiang
BQ
D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D013274:Stomach Neoplasms / Q000378:metabolism / Q000473:pathology*; D014162:Transfection
C411744:DMAP1 protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D012097:Repressor Proteins