Effect Of Promoter Region De Novo Methylation On C-myc And H-ras Expression And The Growth Of Cancer Cells

ObjectiveBy methylating the CpG islands of oncogenes (c-myc and H-ras) promoterregions with S-adenosyl-L-methionin (SAM), to explore the transcription regulationof de novo methylation on oncogenes and the effects on growth of cancer cell lines(MGC803 and HT29) and normal cell line (Chang liver cell line), and to search a newtarget for clinic diagnosis and treatment of cancers.MethodsCultivate the cell lines in vitro. Some cultures, choosed as the test groups T₁,T₂and T₃ on random, were exposed to the methyl donor SAM to methylate the CpGislands of oncogenes promoter regions, and the others were cultivated without SAMas the control groups C₁,C₂ and C₃. Growth speed of the cells was evaluated by thecell growth curves and the inhibition ratios from MTr assay. Abstract the DNA ofwhole genome, and detect methylation status of c-myc and H-ras promoter regions inall the groups by Methylation-specific PCR (MSP). The expression of the oncogenesmRNA was determined by reverse transcription polymerase chain reaction (RT-PCR)and real time RT-PCR using GAPDH as an interior reference, andimmunohistochemistry was used to analyze the difference of the protein expression ofthe oncogenes between the test groups and the control groups.Results1. MTT array: according to the cell growth curves, cancer cells in the test groupsgrew more slowly than those in the control groups; No distinguished differencewas found in the growth speed between the test group and control group of thenormal cell line although the growth of the cells in the test group was inhibitedpartly. Moreover, the inhibition ratios of cancer cell lines (22.0% and 20.3%)conspicuously differed from those of the normal cell line (6.8%) (P＜0.05). 2. MSP: treated with SAM, the CpG islands of the oncogenes promoter regions inMGC803 and HT29 cell lines were methylated and all the cytosines remain ascytosines, while the CpG islands in the control groups untreated with SAM werenot methylated and all the cytosines in CpG dinucleotides had been converted tothymidine; but there is no significant difference between the test group and controlgroup of the normal cell line whose CpG islands were both highly methylated.3. RT-PCR and real time RT-PCR: the mRNA expression of oncogenes c-myc andH-ras were inhibited obviously in the test groups compared to the control groupsin the cancer cell lines (P＜0.05). But there was no significant difference betweenthe test group and control group of the normal cell line.4. Immunohistochemistry: the protein expression of oncogenes c-myc and H-ras wasmuch lower in the test groups than in the control groups as to the cancer cell lines(P＜0.05). But it had no difference between the test group and control group of thenormal cell line.ConclusionThe promoter regions of the oncogenes c-myc and H-ras were hypermethylatedin the normal cells, while they were hypomethylated in the cancer cells, whichsuggested that it could be used as a biological marker and diagnostic target of cancers.SAM could effectively methylate the promoter regions of c-myc and H-ras whichwere hypomethylated in the cancer cells, inhibit their transcription activity, and finallyresult in the depression of the growth speed of the cancer cells, which indicated a newway of the clinic treatment of cancers.