| The enzyme DNA 5-cytosine methyltransferase 1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the 5th position of cytosines residing in the dinucletide sequence CpG. DNA methylation patterns correlate inversely with gene expression, therefore, DNA methylation has been suggested to be an epigenetic determinant of gene expression. This is supported by the recent findings that increased DNA methyltransferase levels are connected with the inactivation of some tumor suppressor genes, DNA repair gene, apoptosis regulating gene of cell cycle, hormone receptor gene and inhibition angiopoesis gene. In addition to its potential role in gene expression, the DNA methyltransferase is also implicated in oncogenesis. Elevated levels of DNA methyltransferase activity have been observed in many cancer cells in vitro and tumors in vivo. RNA interference (RNAi) is the phenomenon in which double-stranded RNA (dsRNA) specifically suppresses the post- transcriptional expression of a gene target. RNAi, a new and rapidly evolving technology too, is already regarded not only as a technique that has great promise for mammalian functional genomics, but also as a therapeutic agent. In this study, we have developed potent shRNAi plasmids corresponding to dnmt1 gene, transfected it into human gastric cancer cell lines AGS, assessed the specificity of shRNA by examining the DNMT1 protein levels and mRNA levels in AGS cells. Our data show that pshRNA-dnmt1 can specifically and efficiently inhibit dnmt1 gene expression. So in the second and third phases of our research, we employed it to study the response of cancer cells in vitro and mouse in vivo. Our results strongly suggest that recombinant plasmid pshRNA-dnmt1 can effectively inhibit the proliferation and induce the apoptosis of human gastric cancer cell lines AGS. Thus, pshRNA-dnmt1 targeting dnmt1 gene may provide an innovative approach to cancer therapy. Objective: To construct the plasmid containing short hairpin RNA (shRNA) of DNA methyltransferase 1(dnmt1), and to suppress the expression of gene dnmt1 in human gastric cancer cell lines AGS. Methods: A 19bp reverse repeated motif targeting coding sequence of dnmt1 with 4bp spacer was synthesized, and inserted into pTZU6+1 to generate the pshRNA-dnmt1 plasmid. Recombinant plasmid pshRNA- dnmt1 was transfected into human gastric cancer cell lines AGS to analyze inhibition of gene dnmt1 expression. Results: The recombinant plasmid pshRNA-dnmt1 was successfully constructed. Relative to control, dnmt1 protein levels of AGS cells after 24h of treatment appeared to reduce (inhibition rate: 28.24%), which of 48h showed significant change (68.54%), which of 72h were dramatically inhibited (81.47%). ShRNA-dnmt1 induced a 21.63% inhibition of dnmt1 mRNA after 24h treatment, 52.97% after 48h, and 72.06% after 72h. Conclusion: The results show that the short hairpin RNA of dnmt1 can efficiently and specifically inhibit the expression of gene dnmt1 in AGS cells. Objective: To study the proliferation and apoptosis of gastric cancer cell lines AGS induced by the recombinant plasmid pshRNA-dnmt1 in vitro. Methods: 1. Recombinant pshRNA-dnmt1 without endotoxin was extracted, and was transfected into AGS cells with Lipofectmaine 2000. 2. The cell survival index was determined by the change of cell morphology under light microscope and MTT study. 3. Cell cycle and apoptosis of AGS cells after transfection were detected with Flowcytometry (FCM). 4. The apoptosis of gastric cancer cell lines AGS was also evaluated by AO/EB staining, electron microscope and TUNEL test. Results: 1. Recombinant pshRNA-dnmt1 without endotoxin was extracted successfully. 2. MTT results showed that the cell survival rates of AGS cells transfected with pshRNA-dnmt1 24h, 48h and 72h respectively were 79.49%, 51.63% and 39.16%. 3. The data of FCM indicated that the growth of AGS cells was directly suppressed: S phase cells reduced dramatically and G2-M phase cells increased significantly. A significant hypodiploid nucleus of peak (the a...
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