Hypermethylation of Let-7 Promoter Is Involved in [cagA.sup.-]Induced Ras Upregulation in Gastric Epithelial Cells

Introduction: Infection with [cagA.sup.-]positive H.pylori is considered to be a risk factor for the development of gastric cancer. The authors investigated the effect of CagA expression on miRNA expression in gastric epithelial cells by miRNA array in vitro, and revealed that let7 was detected as one of the miRNAs whose expression was most significantly affected by CagA expression. The aim of the present study was to determine mechanism by which CagA suppressed let-7 expression. Methods: For in vivo study, CagA transgenic mice of which CagA expression was regulated by tetracycline (Tet)-off system was constructed. Ras and let-7 expression in the stomach were compared between Tet-on and -off mice. To investigate the precise mechanism in vitro, rat gastric mucosal cultured cell line (RGM-1) cells expressing cagA were established by Tet-off system. The effect of the replenishment of decreasing let-7 expression was investigated by let-7 precursor transfection. Promoter CpG island methylation of rat let-7 was ascertained by a PCR-based methylation sensitive restriction enzyme assay. To investigate the mechanism of aberrant hypermethylation in [cagA.sup.-]expressing cells, DNMT1, 3a, and 3b expression were determined by RT-PCR. Results: In the CagA transgenic mice study, CagA expressing stomachs revealed significantly decreased let-7 and enhanced Ras expression compared to the control group. In in vitro study, it was confirmed that CagA significantly reduced let-7 miRNA expression, and induced Ras expression. Let-7 precursor restored accelerated Ras expression in CagA expressing cells. Demethylating agents led to recovery of let-7 expression and suppression of Ras expression. CpG methylation analysis showed that CagA expression induced hypermethylation of rat let-7 promoter region. DNMT3b expression was enhanced in CagA expressing cells comparing with control, whereas no significant difference in DNMT1 or DNMT3a expression was detected between control and CagA expressing cells. Conclusions: The results indicate that CagA is involved in let-7 downregulation through hypermethylation due to DNMT3b induction, leading to its downstream Ras upregulation, which suggests that let-7 downregulation accounts for the [cagA.sup.-]related gastric carcinogenesis.