The Mechanism Of Over-expression P21^waf1/CIP Induce P16^Ink4a Demethylation In ALT Tumor Cells

Backgound:Cellular senescence is an important barrier for anti-cancer.It plays an important role in inhibiting tumorigenesis. Cellular senescence can be avoided accumulation of cell damage, and eliminate the potential threat of tumor formation.Meanwhile, cellular senescence is often considered to be a risk factor in tumor formation.With a constant number of cells in normal tissues, senescence cells increasing can destruct renovation and repairation of damaged tissue, then make the microenvironment of the cells change, affect the structure and function of tissue.lt benefits to formating tumor.Therefore, aging is a double-edged sword,and it is a important significance to research in-depth for anti-aging and cancer treatment.In order to study the relationship between aging and cancer, we have created Werner syndrome (Werner syndrome,WS) mice model in early experiments.The model faithfully reproduce the human Werner syndrome, cancer susceptibility, and other symptoms. With premature aging and cancer susceptibility characteristics, the model is a important tool to study the dialectical relationship between aging and cancer.This is telomerase knockout mouse model.Therefore, it is a tumor with negative telomerase, using the mechanisms of Alternative Lengthening of Telomere to maintain the tumor cell proliferation. We found the MEF(G5) of WS mice with premature aging of cells in early experiments, but some cells escape senescence, form immortalized cell lines(395-3B-1,395-3B-2,395-9B-1,395-6B-1,395-7A-1,395-7A-3).And a few cells(395-3B-1,395-3B-2,395-9B-1)could form tumors in vivo of SCID mice, called ALT.In addition, we found p16 gene promoter region CpG island hypermethylation in these ALT tumor cells at the molecular level, however, when overexpress the exogenous p21 in the cells, the p16 gene promoter demethylation. It shows that some amino acids of histones modificated by acetylation, methylation, phosphorylation and ubiquitination, chromatin structure and DNA methylation affected, which regulate gene expression. Additionally, some studies have shown that miRNAs can regulate the DNMTs expression. Indirectly, it can affect the downstream target gene promoter methylation and expression, and participate in the progression of disease. Therefore, it is a certain significance to study the mechanisms of p21 inducing p16 demethylation for cancer and the related diseases.Objective:Explore the machinery of overexpress the exogenous p21 induce the p16 gene promoter demethylation in ALT tumor cells.Methods:In order to validate the authenticity of early experiments,we detected the related gene and protein expression after overexpression of exogenous p21 in 395-3B-2 tumor cells by Western-Blot and Real-Time PCR assay,and,detected p16 methylation status by BSP.In addition,we detected the relative of miRNAs expression after overexpression p21 in these cells by Real-Time PCR assay.To makesure that the phenomena associated with miRNAs.We established Dicer knockdown vector by RNA interfere, trying to restrain miRNAs formation to verify miRNAs involved from the reverse side.Results:We found the p16 promoter region of histone modification changes by chromatin immunoprecipitation and Western blot,the histone modifications of transcription inhibition reduced, the histone modification of transcriptional activation increased in early experiments.We found the Ezh2 and Bmi-1 which are the members of polycomb family were lower significantly.We found the p300 which is the transcriptional co-activator of DNMT1 down-regulation byWestern-blot.Therefore,we deduced that overexpression p21 inhabit the DNMT1 transcription by reducd the p300 expression.We found that expression of DNMT1 decreased to make the p16 demethylation after overexpression exogenous p21 in 395-3B-2 ALT tumor cells.In addition,we found the miR-152 and miR-185 which can directly bind the 3’UTR of DNMT1 mRNA and inhabit DNMT1 expession were decreased.The reverse experiments proved that miR-185 and miR-152 are involved in the regulation of overexpression p21 indeced p16 demethlation.However,we found that Dicer knockdown can make Akt protein phosphorylation in the process of researching Dicer knockdown to restrain miRNAs.The Phospho-Akt protein can make P21 protein phosphorylation,p21 can’t enter into the nucleus.Therefore, it is lack of evidence to validate miRNAs participation in p21 induces p16 demethylation by knocking down the Dicer.Conclusions:To sum up, overexpression of p21 in ALT tumor cells can induce demethylation of the pl6 promoter by p300 expression down and DNMT1 down-regulation. In addition, overexpression of p21 leads to up-regulation of miR-152 and miR-185 in the cell. They can directly into the 3’UTR of DNMT1 and inhibit the expression of DNMT1, leads to demethylation of the p16 promoter region.