Effects Of 5-aza-2'-deoxycitydine And Trichostatin A On Expression And Methylation Of P16 And HMLH-1 Gene In Human Gastric Cancer Cell Lines

ObjectiveRecently ,Many reserchs showed transcriptional silencing of many tumor-supressing gene for promoter hypermethylation of tumor- suppressing is a well-recognized mechanism the activation of oncogenes or inactivation of tumor suppressor gene is one of the key steps in the mocecular process of tumorigensis.it has been popular presumed the abnormal methylation is the third mechanism except abnormal and mutation Which plays an important role in tumorigenesis and developmentGastric carcinoma is one of the most common malignant tumor in the world and the most popular malignant tumor in china .Over the past few years,the five-year survival rate of early gastric cancer has been enhanced. But it has been not improved for advanced gastric cancer.It was demonstrated that the carcinogenesis of grastric carcinoma is a multi-step process participated with polygene.Methylation-specificPolymeraseChainReaction(MSP)andReverseTranscription-Po ly-merase-c-hainReaction(RT-PCR) were used in the experiment to investigate the effect of 5-aza-2'-deoxycitydine(5-Aza-dC) and Trichostatin A(TSA) on expression and methylation of P16and hMLH-1 geng in gastric carcinoma cell.It will be helpful to reveal the regulation of cell proliferation,differentiation and cancerization with the study on this aspect. Furthermore it will probably provide theoretical and experimental evidence to clinical diagnosis,treatment and prognosis of tumors Methods1 Cell were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 100×10³ U/L penicillin ,100×10³ U/L Streptomycin and incubate in 5%CO₂ at37℃.Divid cell line into four group and them was treated by 5-aza-2'-deoxycitydine (5-Aza-dC) and Trichostatin A(TSA).(1)5-Aza-dC (5μmol/L)was used for 72 h in the treatment.(2) TSA(300 nmol/L) was used for only 24 h in the treatment.(3)5-Aza-dC was used for 48 h followed by TSA for an additional 24 h in the combined treatment.2 Genomic DNA was extracted from gastric cancer cell line by the Phenol-Chloroform method; Extracted whole RNA by using TRIZOL.3 Methylation-specific Polymerase Chain Reaction(MSP):Genomic DNA was denatured by NaoH and modified by sodium bisulfite.DNA sample were then purfied using Wizad DNA clean-up (PROMEGA)and the performed PCR by using unmethylated primer and methylated primer.4 Reverse Transcription-Poly-merase-chain Reaction : whole RNA was synthesizedcDNA.PCR was performed by use P16 and hMLH-1 gene primers and GAPDH RT-PCR was carried simultaneously .5 PCR products were separated by electrophoresis .Analyzed the electrophoretic images by Fluor Chen V.2.0 software.The experiment was repeated three times .Results1. The two cell lines showed a characteristic DNA methylation status in each promoter region of P16 gene and hMLH-1 gene . P16 gene was hypermethylated in MGC-803 and MKN-45; hMLH-1 gene was hypermethylated in MGC-803,but Hemi-methylated in MKN-452. P16 and hMLH-1 mRNA was expressed in MGC-803 and MKN-45 gastric cancer cell lines after 5-Aza-dC and TSA treatment,but it was undetectable or express weakly before the treatment.3. The methylation of P16and hMLH-1 gene in MKN-45 and MGC-803 cells was reversed after 5-Aza-CdR and TSA treatment. ConclusionAberrant methylation of P16 and hMLH-1gene is a common event in the occurrence and progression of gastric cancer.The methylation of promoter region in CPG island is a main causal of P16 and hMLH-1gene transcriptional inactivation, Treat with5-Aza-dC and the combination of 5-Aza-dC and TSA effect is similar, Dramatically enhance experession of mRNA in human gastric cancinoma cell. DNA methylation and experession of mRNA was not affected after TSA treatment in human gastric cancinoma cell...