Molecular Mechanism Of Over-expressing P21^Waf1/Cip1Induced P16^Ink4aDemethylation In ALT Tumor Cells

Cell senescence is a double-edged sword for the tumor:on the one hand,cell senescence can remove cells with damages or with abnormal oncogene activation to prevent carcinogenesis;on the other hand,aging cells facilitate the occurrence of adverse mutations,telomere dysfunction causes genome disorders,and the microenvironment surrounding aging cells has been changed,all those could promote the occurrence of tumors.Thus,understanding the relationship between aging and cancer is very important for anti-aging and tumor prevention and treatment.Werner syndrome(WS)is a classic model for studying the relationship between aging and tumor.Since the telomerase gene is knocked out in the WS mouse model,the tumor in this mouse is telomerase-independent,and using Alternative Lengthening of Telomere(ALT)mechanism to maintain telomere length and function.Previous studies have found that the G5 MEF cells from WS mice are prone to senescence,and part of the cells escaped from senescence to form immortalized cell lines(395-3B-1,395-3B-2,395-9B-1,395-6B-1,395-7A-1,395-7A-3).Among them 395-3B-1,395-3B-2,395-9B-1 were capable of forming ALT tumors in SCID mice.The CpG islands of the p16 promoter region in these ALT tumor cells were highly methylated.However,when exogenous p21 was overexpressed in these cells,the expression of DNMT1 is significantly reduced,leading to p16 re-expression,induced tumor cells into the aging state.It was also found that p21 induced the expression of miR-185 and miR-152,which could directly target DNMT1 mRNAand inhibit its expression,but the mechanism was not clear.Based on previous results,this study included two parts:the first part is to further study the molecular pathway of exogenous overexqpressing p21 regulated DNMT1;the second part is to further study the effects of miR-185 and miR-152 on p16 methylation.The 395-9B-1 cells was used and the p21 was overexpressed in this cell.The expression of Spl and E2F1,DNMT1 transcription factors,were detected by Western blot.The expression of Spl and E2F1 was significantly reduced by p21 overexpression.This result suggested that p21 might inhibit the expression of DNMT1 by regulating the expression of it's transcription factors Sp1 and E2F1.It is well known that p21,as an important cell cycle suppressor,can inhibit the release of E2F by inhibition of cyclin kinases.As a transcription factor,E2F is involved in the regulation of multiple gene expression.We found that overexpressing p21 in the ALT tumor cells could directly inhibit E2F1 expression,suggested that p21 is not only involved in the functional regulation of E2F,but also involved in the expression regulation of E2F1.On the other hand,we transfected miR-185,miR-152 mimics in 395-9B-1 cells to further study the effects of those two miRNAs on the cells.We found that both miRNAs significantly reduced the expression of DNMT1 and that the decrease in DNMT1 protein levels was more pronounced with respect to mRNA levels.And thus induced demethylation of p16,resulting in p16 re-expression.These data suggested that miR-185,miR-152 might regulate DNMT1 expression mainly through at the protein level,such as translational regulation or protein degradation,as well as at its transcrption level.In order to further understand the mechanism of miR-185 and miR-152 on p16 methylation,the expression level of H3K4me3 and H3K27me3 was detected.The results showed that miR-185 and miR-152 overexpressing could significantly up-regulate the expression of H3K4me3.Accordingly,we hypothesized that theup-regulation of H3K4me3 could inhibit the binding of DNMT3L to nucleosomes and further inhibit the methylation of DNMT3A and DNMT3B,leadiIng to desmethylation of p16,which led to the re-expression of p16.We found that co-transfection of miR-185 and miR-152 could inhibit the of trimethylation of H3K27.Whereas this was not found in cells transfected with miR-185 or miR-152 alone.This result suggests that there is a synergistic effect between miR-185 and miR-152 that inhibits the expression of H3K27me3 and relieves transcriptional inhibition of p16.In addition,the expression of p21 protein showed that miR-185 and miR-152 had a certain feedback on p21 protein expression.The p16.p21 are important cell cycle inhibitory factors,we carry out cell cycle detection of these cells.In our expectation,p21 and p16 were elevated in cells when those miRNA mimics were overexpressed,but the S phase was significantly increased and the cell cycle was accelerated.In order to explain this phenomenon,we detected cyclin kinase CDK4,CDK6 and found that both cyclin kinases increased to a certain extent in protein levels.This phenomenon needs to be confirmed by further studies.In addition,we accidentally discovered that cationic liposome transfection reagent lipo2000 affects the expression of DNMTl protein in 395-9B-1 cells.In order to further study the effect of lipo2000 on the expression of DNMT1 in cells,we used Hela cells for further validation.The results showed that lipo2000 treatment inhibited the expression of DNMT1 in Hela cells at the post-transcriptional level,while the Hela cells restored the protein expression of DNMT1 by enhancing its transcrption level later.And different concentration of lipo2000 showed different impact on regulating DNMT1 expression.Compared with high concentration lipo2000 treatment,the effect of low concentration of lipo2000 in the expression of DNMTI was more obvious,but the duration was relatively short.In conclusion,our data show that overexpression of p21 in ALT tumor 395-9B-1 cells inhibits the expression of DNMT1 by downregulating transcription factors Sp1,E2F1.Overexpression of miR-185 and miR-152 can also inhibit the expression of DNMT1.And miR-185,miR-152 can further inhibit the methylation function of DNMT3Aand DNMT3B by up-regulating the expression of H3K4me3.At the same time there is a synergistic effect between miR-185 and miR-152 that can inhibit the trimethylation of H3K27(H3K27me3)modification and further promote p16 transcription.In addition,miR-185,miR-152 can also up-regulated CDK4,CDK6 expression,suggested that one kind of miRNA may play a role in multiple pathways and multiple life activities,and there may be complex interactions between miRNAs.As an important tumor suppressor gene in the process of tumor development,p16 often manifested as hyperimethylation and inactivated.In this study,we found that p21 and miR-185,miR-152 play an important role in the regulation of p16 epigenetic modification and provide anew sight for tumor control.