[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. CONCLUSION: 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.

[Effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS].

BACKGROUND & OBJECTIVE: Dnmt1, a major DNA methyltransferase gene, is highly expressed in many cancers and lowly expressed in normal adult cells, therefore, its overexpression is closely related to tumorigenesis. This study was to assess effects of dnmt1 gene silencing on cell cycle, proliferation, and apoptosis of gastric cancer cell line AGS. METHODS: The eukaryotic expression plasmid pshRNA-dnmt1, containing the sequence of short hairpin RNA (shRNA) targeting dnmt1, was constructed and transfected into AGS cells. PBS-treated cells and pTZU6+1-transfected cells were set as control. The expression levels of dnmt1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell cycle was analyzed by flow cytometry; cell survival was analyzed by MTT assay; cell apoptosis was evaluated by AO/EB double staining, electron microscopy, and TUNEL. RESULTS: pshRNA-dnmt1 targeting dnmt1 was successfully constructed, and confirmed by sequencing. Relative to control, 24, 48, and 72 h after transfection of pshRNA-dnmt1, the inhibitory rates of dnmt1 protein levels in AGS cells were 28.24%, 68.54%, and 81.47%, respectively, and those of dnmt1 mRNA levels were 21.63%, 52.97%, and 72.06%, respectively. The growth of AGS cells was suppressed 72 h after transfection: S phase cells were reduced from (36.58+/-1.76)% to (18.54+/-6.59)% (P<0.05), and G(2)/M phase cells were increased from (6.18+/-0.32)% to (18.53+/-1.42)% (P<0.05). Cell survival rates were 79.49%, 51.63%, and 39.16%, respectively, 24, 48, and 72 h after transfection of pshRNA-dnmt1. A lot of apoptotic and necrosis cells were observed after transfection. CONCLUSIONS: The recombinant plasmid pshRNA-dnmt1 can efficiently and specifically inhibit the expression of dnmt1 gene and the proliferation of AGS cells, and induce cell apoptosis. It provides evidence for gene therapy of human cancers.

Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori.

AIM: To construct a recombinant E. coli strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori (H pylori). METHODS: The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E. coli strain. This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis. Finally this sequence AF479754 was compared with HP0638 and JHP0581. RESULTS: The sequence of the aim gene was obtained. It had 924 base pairs. The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581. CONCLUSION: Although the aim gene was obtained, but it was different from the published sequence of GenBank. It is not clear what makes this difference. Maybe it is because different strain was used or because there were some variations. So more researches are required to prove it.