关键词: 胃癌, DNA甲基转移酶, 干扰, 甲基化

Abstract:

AIM: To inhibit the expression of DNMT1 (DNA metylation transferase 1, DNMT1) gene in gastric cancer BGC-823 cells by RNAi and to investigate the expression of p16 gene after the silence of DNMT1.  METHODS: siRNA plasmid 1,2,3 and negative control siRNA plasmid b were constructed according to the nucleotide sequence of DNMT1. Gastric cancer BGC-823 cells were transfected with the constructed siRNA plasmid in mediation of liposome. Transcription levels of DNMT1 mRNA were determined by Q-PCR, protein expression levels by Western blot to select the best interference plasmid after the DNMT1 gene silence. Then used the same methods to investigate the expression of p16 gene in gastric cancer cells, and effects of inhibiting the expression of DNMT1 on the p16 gene of gastric cancer cells. RESULTS: The RNAi could effectively inhibit the expression of DNMT1 gene in gastric cancer BGC-823 cells. After transfection, transcription levels of DNMT1 mRNA were determined by Q-PCR, and translation levels of DNMT1 protein by Western blot. The results showed that the expression of DNMT1 in siRNA plasmid 2 group was markedly down-regulated compared to other transfection groups, negative control group and blank control group (P＜0.05); so siRNA plasmid 2 was the best interference plasmid. Used the same methods to investigate the expression of p16 gene in the best interference group (siRNA plasmid 2), negative control group and blank control group, the expression of p16 gene mRNA (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). CONCLUSION: RNAi can inhibit the DNMT1 gene expression to promote the de-methylation and restore expression of  p16 gene.

Key words: gastric cancer, DNA methylation transferase, RNA interference, methylation