The Effects And Mechanism Of Different Types Of CagA~+ Helicobacter Pylori On Expression Of Cx32 And Cx43 MRNA In GES-1 Cells

Objective To observe the effects of the East Asian CagA+H.pylori and Western CagA+H.pylori on expression of Cx32,Cx43 mRNA in GES-1 cells, and to investigate the mechanism related to mRNA expression change by the methylation pattern of gene promoter CpG island.Methods The East Asian CagA+H.pylori and Western CagA+H.pylori were isolated from clinical patients, we co-cultured these two types H.pylori with GES-1 cells for 24 hours,48 hours and 72 hours, with bacteria/cells ratio of 100:1 respectively. The expression of Cx32 and Cx43 mRNA were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR(qPCR) methods, while the methylation pattern of Cx32 gene promoter CpG island was detected by methylation-specific PCR(MSP) method. Because the Cx43 gene promoter CpG island was not found in our study, so we did not detect its methylation pattern.Results1.In control group,the number of cells was increased with time, cells had normal shape, like polygon or rhombus,arranged in order and clear boundary. In the East Asian CagA+H.pylori and Western CagA+H.pylori groups, the number of cells also increased with time, but compared with control group,the number of cells was decreased, cells became elongated, some of them like "hummingbird " phenotype, had indistinct cells borders and disordered arrangement.2.In control group, the expression of Cx32 and Cx43 mRNA was increased with time;While in the East Asian CagA+H.pylori and Western CagA+H.pylori groups, both the expression of Cx32 and Cx43 mRNA were decreased with time,and lower than control group at 24 hours,48 hours and 72 hours(P< 0.05).Especially for the East Asian CagA+H.pylori group,the expression of Cx32 and Cx43 mRNA was lower than the Western group at each time point (P<0.05)3. In control group,the methylation pattern of Cx32 gene promoter CpG island was non-methylated at 0 hours,24 hours,48 hours and 72 hours; Both the East Asian and Western CagA+H.pylori group were non-methylated at 0 hours, and hemi-methylated at 24 hours,48 hours and 72 hours.ConclusionBoth the East Asian CagA+H.pylori and Western CagA+H.pylori, especially with the East Asian CagA+H.pylori can down-regulate the Cx32 and Cx43 mRNA expression,this down-regulation is depend on time.The mechanism of the Cx32 mRNA expression down-regulated by the East Asian CagA+H.pylori and Western CagA+H.pylori may be relate to the methylation pattern of Cx32 promoter CpG island.