Inhibition Of The Expression Of DNMT1 To Effects On The P16 Gene Of Gastric Cancer Cells

Objective: To inhibit the expression of DNMT1(human DNA metylation transferase 1,h DNMT1)gene in gastric cancer BGC-823 cells by RNAi and investigate the expression of p16 gene in gastric cancer BGC-823 cells after the silence of DNMT1.Methods: sh RNA sequence was designed and synthesized by Gene synthesis company according to the nucleotide sequence of h DNMT1 m RNA reported in Gen Bank, used the sh RNA template for construction of si RNA plasmid 1,2,3 and negative control si RNA plasmid b. Cultivation of E coli which containing plasmid, measure the plasmid concentration and use the plasmid DNA gel electrophoresis to confirm the size of plasmid after plasmid extraction. Gastric cancer BGC-823 cells were cultivated in 6-well plate and the cells were transfected with the constructed si RNA plasmid in mediation of liposome next day,used the Fluorescence microscope to investigate transfection efficiency after the Gastric cancer cells were cultured for 48-72 hours and collected the High efficiency cells to use in the subsequent experiments,The experiment were divided into transfection group(si RNA plasmid 1,2,3) 、 Negative control group si RNA plasmid b and blank control group 0. Determined for transcription levels of DNMT1 m RNA by Q-PCR,for Protein expression levels by western-blot, selected the best interference plasmid after the DNMT1 gene silence. Then the experiment were Divided into The best interference group、negative control group and blank control group, used the same methods to investigate the expression of p16 gene ingastric cancer cells, and this way can test inhibition of the expression of DNMT1 to effects on the p16 gene of gastric cancer cells.Results: The RNAi can effectively inhibit the expression of DNMT1 gene in Gastric cancer BGC-823 cells. After transfection, determined for transcription levels of DNMT1 m RNA by Q-PCR, The results showed that the expression of DNMT1 m RNA in si RNA plasmid 2 group(0.3253±0.0301) was markedly down-regulated compared to other Transfection groups 、 negative control group( 1.0087±0.1584) and blank control group(1.8576±0.5603), after the SPSS17.0 calculation, P < 0.05; si RNA plasmid 1 group(0.7095±0.1154) was compared to si RNA plasmid 3 group(0.6163±0.1110), P > 0.05; all Transfection groups were down-regulated compared to negative control group and blank control group, P<0.05; negative control group was down-regulated compared to blank control group, P<0.05. Determined for translation levels of DNMT1 protein by western-blot, the expression of DNMT1 protein in si RNA plasmid 1 group(0.6772±0.0203) 、 si RNA plasmid 2 group(0.5114±0.3866) and si RNA plasmid 3 group(0.6218±0.0304), the results was similar to the Q-PCR, negative control group(0.8135±0.0224) was compared to blank control group(0.8164±0.0734), P>0.05.So si RNA plasmid 2 is the best interference plasmid. Determined for transcription levels of p16 m RNA by Q-PCR, for translation expression levels by western-blot in The best interference group(si RNA plasmid 2)、negative control group and blank control group, the expression of p16 gene m RNA(1.6727±0.2242) and protein(0.9227±0.0337) in the best interference group was markedly up-regulated compared to the negative control group(1.0025±0.0877) 、(0.5440±0.0229) and blank control group(0.4729±0.0940)、(0.4767±0.0774), P<0.05; the expression of p16 protein in negative control group was compared to blank control group, P>0.05.Conclusion: The DNMT1 gene-induced abnormal methylation of promoter region of cancer suppressor gene p16 was a key factor for onset and progress of gastric cancer, and RNAi can inhibit the DNMT1 gene expression to promote the demethylation and Restore express of p16 gene which can inhibit onset and progress of gastric cancer and provide a potential route for therapy of gastric cancer.