Transcription And Expression Study Of Promoter Region Demethylation On CKIs Genes In Human Colorectal Cancer Cell

Colorectal cancer (CRC) is one of the most common malignant tumors, its morbidity has raised to the third cause in recent years in China. A great number of evidences improved that various factors and mechanisms are involved in CRC carcinogenesis. From the normal intestinal epithelial cell to intestinal cancer must undergo a series of process which including hyperplasia, early-stage adenoma, middle -stage adenoma, late-stage adenoma, in situ carcinoma and metastatic carcinoma, in which involves activation of oncogenes and inactivation of tumor suppressor genes. It have been demonstrated that the mutation of adenomatous polyposis coli (APC) and MMC in 5q chromosome, the mutation of K-ras gene in 12p, deletion and/or mutation of DCC in 18q and p53 deletion in 17p were at least altered. These multistep and multifactorial accumulation had played an important role in CRC carcinogenesis.However, these changes of oncogene and tumor suppressor gene could not completely explained the activation/ inactivation by the classical regulatory control (two-hit hypothesis). Recent data suggested that CRC appears to be a process that isfuelled by genetic alterations and by epigenetics mechanism.Epigenetics refer to the study of expression of the affected genes that can be mitotically inherited, including modifing the gene transcription and translation, but is not associated with the changes in the coding sequence of the affected genes. It manily involves DNA methylation and histone deacetylation.DNA methylation, the most important mechanism in epigenetics, is a covalent modification of the fifth carbon position of the pyrimidine ring of cytosines in CpG dinucleotides and DNA methyltranferase (DNMTs) catalyzes the transfer of a methyl residue from S-adenosyl-methionine (SAM) to a cytosine of CpG sequence. DNA hypermethylation is associated with transcriptional inactivation of including mismatch repair gene, vascular regulatory gene, defined tumor suppressor genes, adhesion molecule gene in human cancer and is established as the third mechanism by which tumor suppressor genes are inactivated. Among them, the promoter region hypermethylation of cyclin-dependent kinases inhibitors are confined play an important role in cell cycle uncontrol and in CRC carcinogenesis.Cell cycle is a complex series of phenomena, occurring between the end of one cell division and the end of the next, by which cellular material is divided between daughter cells. Cell cycle are divided to four phases: G1, S, G2 and/or M phase and regulatored by three factors, which including cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinases inhibitors (CKIs). CKIs is a kind of important tumor supressor gene, which is classified to two groups, inhibitor of CDK4 (INK4 ) and INK-interacting protein/kinase inhibition protein (CIP/KIP). CKIs has important negative effects on cell cycle regulation, cell over proliferation. So far, methylation of CKIs CpG promoter islands has been described as s mechanism of gene silencing.Therefore, in this paper, we are to explore the transcription and expression effects of promoter region demethylation on CKIs genes in human colorectal cancerRKO cell in vitro by exposed to the specific demethylation agent--5-Aza-2'-deoxycytidine (5-Aza-CdR) , and to detect the methylation status ofpromoter region of CKIs, and to evaluate the relationship between methylation status of promoter 5' CpG island and the biological behavious in human CRC cells and 5-Aza-CdR effect on CRC induced by DMH in mouse (in vivo).Part I The detection of methylation status of CKIs promoter region in RKO cells in vitroMethylation-specific PCR (MSP), T-A coining and DNA sequencing were used to detect the methylation status of CKIs including INK4 (p15ink4b and pl6ink4a/CDKN2) and CIP/KIP (p21/cip and p27/kip) promoter region in RKO cells respectively.The results revealed that the INK4 (p15ink4b and pl6ink4a/CDKN2) promoter region are in DNAabrreant hypermetrhylation in RKO cells and the CIP/KIP (p21...