The Effects Of MNNG On The Telomerase Of Human Gastric Epithelium Resulting From Methylation Regulation

MNNG(N-methyl-N'-nitro-N-nitrosoguanidine)is a direct acting carcinogen. Animal experiments have shown that MNNG induces gastric cancer, but the mechanism of carcinogenesis has been unclear.In addition to carcinogenesis,the mutagen are found to inhibit cellular proliferation,induce apotosis,enhance immune function,and so on.Therefore, the effects of mutagenic substance on human are complicated. Telomerase is the ribonucleoprotein complex and is reactivated in most tumors. Telomerase activity is tightly regulated by expression of the human telomerase reverse transcriptase(hTERT), the telomerase catalytic subunit gene. The expression pattern of hTERT is correlated with methylation of the hTERT promoter.Based on the above findings, I have this study to investigate the effects of MNNG on exression of telomerase and hTERT in human gastric epithelial cells and analyze the methylation status of the promoter region of the hTERT after MNNG exposure. The methods and results are following.1. Isolation, culture and identification of human gastric epithelial cells from the gastric mucosa obtained by routine operation.The gastric epithelial cells were dissociated by stirring the minced tissue in collagenase/dispase solution with a magnetic stirrer. The viability of cells had a maximal increase and reached the apogee on the 4th day, 33.3% of the cells had entered S phase following exposure BrdU. Almost all the cells contained PAS positive granules with high inoculum density and stained positively using a CK-18 antibody. On transmission electron microscopy, epithelial characteristics such as microvilluslike projections,secretary granules and junctional complex were observed.2. The effects of MNNG on the expression of telomerase and hTERT of human gastric epithelium Telomerase activity was assessed with telomeric repeat amplification protocol (TRAP). Expression of hTERT was observed by semi-quantity reverse transcriptase polymerase chain reaction(sqRT-PCR), western blot and immunofluorescence. The same as SGC-7901 cells, telomerase was expressed in normal human gastric epithelium. The telomerase activity was decreased after exposure to 6.8uM and 68uM MNNG. The changes of hTERT mRNA and hTERT protein were at equal pace with telomerase after MNNG exposure.3. DNA methylation analysis of the promoter region of the hTERT in normal human gastric epithelium after MNNG exposureUsing methylation specific-PCR(MSP) and bisulfite sequencing PCR (BSP), the methylation status of hTERT CpG island was examined.The same as in SGC-7901, the target sequence contained all methylatable CpGs in normal human gastric epithelium without MNNG exposure .Almost all the CpGs demehtylated after MNNG exposure. In the region, only two binding motifs for myeloid-specific zinc finger protein 2(MZF-2) were retrieved from literatures. The presence of methylation had importance of inhibition for binding between MZF-2 and DNA.The results of this study imply:â‘ A method of primary culture of human gastric mucosal cells has been successfully developed, which allows isolation of numbers of human gastric epithelial cells with high purity.â‘¡Telomerase and hTERT are all expressed in normal human gastric mucosal cells,which inhibited by MNNG.â‘¢The expression of hTERT is positively correlated to methylation of the promoter region of the hTERT. The demethylation of the promoter region of the hTERT is happened after MNNG exposure, which leads to the inhibition of expression of telomerase and hTERT.â‘£MZF-2 is a negative transcription factor. The presence of methylation has importance of inhibition for binding between MZF-2 and DNA. The demethylation induced by MNNG in the promoter region nearby binding motifs for MZF-2 maybe relieve the inhibition and down-regulate of hTERT transcription and telomerase activity.