The Relationship Between Epigenetic Modification And Transcription Inhibition Of P33ING1b In Colorectal Cancer Cell Lines

The first part:The relationship between p33ING1b gene expression and pattern of epigenetic modificationObjectiveING1 is a candidate tumor suppressor gene discovered in 1996. Recent research demonstrated that the expression of p33ING1b mRNA was down-regulated in many human tumors, but the mechanisms was not clear yet. This study was designed to make a initial investigate for the mechanisms of the p33ING1b transcription inhibition in colorectal cancer through analyzing the relationship between epigenetic pattern and genetic expression in the term of methylation and acetylation of p33ING1b promoter.MethodsFour colorectal cancer cell lines(HT29, LOVO, HCT116, COLO205)were cultured in vitro and p33ING1b mRNA expression was detected by the real-time quantitative reverse transcription-polymerase chain reaction. By using nest methylation-specific PCR (nMSP),the pattern of p33ING1b promoter methylation was analyzed and the pattern of p33ING1b acetylation was detected by chromatin immunoprecipitation(ChIP) and p33ING1b protein expression was detected by Western Blot.Finally the relationship between the pattern of p33ING1b epigenetic modification and gene expression in different colorectal cancer cell lines was analyzed.Results1.Variable expression levels of p33ING1b mRNA could be detected in colorectal cancer cells of HT29, LOVO, COLO205 and HCT116. Compared with HCT116, the expression level of p33ING1b mRNA in HT29 and LOVO was slightly higher (P>0.05), while that in COLO205 was higher than in HT29 and LOVO (P<0.01).2.Semi-methylation of p33ING1b promoter could be detected in colorectal cancer cell lines of HT29,LOVO,and hypermethylation in HCT116,while unmethylation was showed in COLO205.3.The detecting results of acetylation level of two fragments of histone H3 on the front part and the rear of the promoter in the colorectal cancer cells of HT29,LOVO,COLO205 and HCT116 were similar(r=0.997).The acetylation levels in the four cell lines were different. Compared with HCT116,the acetylation levels in HT29 and LOVO were higher(P<0.05),and the level of COLO205 was the highest(P<0.01).4.Variable protein expression levels of p33ING1b were detected in colorectal cancer cells of HT29, LOVO, COLO205 and HCT116.But the protein expression level of p33ING1b in HCT116 was lower,while that in LOVO and HT29 increased significantly, and that in COLO205 was higher (P <0.05).The protein expression levels of p33ING1b among HT29, LOVO and COLO205 had no statistical difference (P> 0.05).5.The levels of histone H3 acetylation and mRNA expression of p33ING1b in unmethylatied cell lines COLO205 were higher relatively among HT29,LOVO,COLO205 and HCT116,also was protein expression,while that of hypermethylated cell lines HCT116 was lower.The levels of histone H3 acetylation and mRNA expression in semi-methylated cell lines HT29 and LOVO were at the medium level,but their protein expression level of p33ING1b maintained at a higher level.The p33ING1b protein expression had a moderate to high correlation with the H3 acetylation and mRNA expression.Conclusion1.Varible low expression levels of p33ING1b mRNA existed in human colorectal cancer cell lines,and it was positive correlation with protein expression levels,which suggested that there were intimate correlation between silencc of p33ING1b and the occurrence and development of colorectal cancer.2. There were varible levels of hypermethylation and deacetylation of p33ING1b promoter in human colorectal cancer cell lines, which was revealed that hypermethylation and deacetylation of p33ING1b involved in the occurrence and development of colorectal cancer, and promoter methylation and acetylation of p33ING1b were frequent incidents of colorectal cancer.3.Varible low expression levels of p33ING1b mRNA in human colorectal cancer cell lines were negatively correlated with methylation of the gene promoter,and that were positively correlated with the level of gene promoter acetylation,which suggested that the down-regulation of p33ING1b mRNA gene expression in human colorectal cancer cell lines had intimate correlation with methylation and acetylation of p33ING1b gene promoter.4.Hypermethylation and deacetylation of p33ING1b promoter is one of the reasons that cause p33ING1b transcription inhibition of human colorectal cancer cell lines,but other mechanisms may exist,such as other epigenetic modification,or p33ING1b transcription inhibition which was caused by abnormal change of transcriptional regulatory mechanism in the upstream. The second part:The influence of intervention on epigenetic modification of colorectal cancer cell lines and p33ING1b expressionObjectiveThe previous reseach showed that tumor suppressor gene ING1 was closely related to colorectal cancer.The expression of transcriptional spliceosome of p33ING1b mRNA was down-regulated in colorectal cancer,and the pattern of hypermethylation and deacetylation was detected,but its mechanism was not clear yet.This study was designed to make a further investigation for the epigenetic mechanism of the p33ING1b transcription inhibition through the external intervention and analyzed the relationship between the change of epigenetic pattern and genetic expression in p33ING1b promoter.MethodsColorectal cancer cell lines were cultured in vitro and divided into four groups:control group treated without any drug,and the other three experimental groups treated with TSA(TSA group),5-Aza-2'-dc (Aza group),5-Aza-2'-dc +TSA(Aza+TSA group) seperately.The expressions of p33ING1b mRNA were detected by real-time quantitative RT-PCR.The pattern of p33ING1b promoter methylation were analyzed by nMSP.Acetylation levels of p33ING1b fragment 1 and 2 were estimated by ChIP.p33ING1b protein expression was detected by Western Blot.Finally the relationship between the change of p33ING1b epigenetic modification and gene expression in different colorectal cancer cell lines was analyzed,in order to investigate a further mechanism between the p33ING1b epigenetic modification and gene silencing in colorectal cancer Results1.Compared with the control group respectively,group-TSA or 5-Aza-2'-dc alone could up-regulate the expression levels of p33ING1b mRNA in HT29, LOVO and HCT116 slightly(P<0.05),but combined-using of 5-Aza-2'-dc+TSA can up-regulate their p33ING1b mRNA expression levels significantly(P<0.01). Using TSA or 5-Aza-2'-dc alone could up-regulate the expression level of p33ING1b mRNA in COLO205 slightly,but there was no statistical difference (P>0.05).The combined 5-Aza-2'-dc and TSA could up-regulate the p33ING1b mRNA expression of COLO205(P<0.05).2. 5-Aza-2'-dc and the combined 5-Aza-2'-dc and TSA resulted in de methylation which was associated with hypermethylation and Semi-methylation of p33ING1b promoter in colorectal cancer cell lines of HCT116 and HT29,LOVO.In the contrast, TSA alone did not reverse the p33ING1b promoter methylation pattern.3.The effect of intervention to p33ING1b fragment 1 was similar with fragment 2.Using TSA alone could up-regulate acetylation levels of p33ING1b histone H3 in the colorectal cancer cell lines of HT29,LOVO,HCT116 and COLO205(P<0.05),while using 5-Aza-2'-dc alone could up-regulate acetylation levels in them slightly (P>0.05) and no effect on COLO205.The combined 5-Aza-2'-dc and TSA resulted in up-regulating acetylation levels in them significantly(P<0.01).4.Protein expression of p33ING1b increased after intervention of using TSA or 5-Aza-2'-dc in the colorectal cancer cell lines of HT29,LOVO and HCT116(P<0.01).The combined 5-Aza-2'-dc and TSA could enhance protein expression of p33ING1b(P <0.05),and the effect on HCT116 was maximal.5.Acetylation,mRNA and protein expression in p33ING1b were heightened by intervention to colorectal cancer cell lines.Especially in methylated cell lines of HCT116,methylation could not be reversed after using TSA alone,and the levels of acetylation,mRNA and protein expression in p33ING1b were elevated slightly (P<0.05).Then using 5-Aza-2'-dc alone,the methylation had been reversed but the levels of mRNA and protein expression in p33ING1b were also mildly elevated (P < 0.05),but acetylation level was not increased(P >0.05).While it was intervened by 5-Aza-2'-dc and then by TSA,its methylation had been reversed quickly and the levels of acetylation,mRNA and protein expression in p33ING1b raised significantly(P<0.01).The effect of intervention to cell growth inhibition was significant.Conclusion1.The hypermethylation of CpG islands in p33ING1b promoter in human colorectal cancer cell lines was associated with 33ING1b silencing.The methyl- transferase inhibitor,5-Aza-2'-dc could help the p33ING1b demethylated, also can up-regulate the p33ING1b expression.2.The acetylation level of p33ING1b in human colorectal cancer cell lines had a negatively correlation with DNA methylation.The histone deacetylase inhibitor,TSA could up-regulate the level of acetylation of p33ING1b, and partly antagonize the silence of p33ING1b which was generated from p33ING1b promoter hypermethylation.3.Regarding to cells HCT116,whose p33ING1b promoter was hypermethylation,if histone deacetyltransferase,TSA is used alone the up-regulation of gene expression would be not obvious,but if the methyltransferase inhibitor,5-Aza-2'-dc is used to make p33ING1b obtain mild re-expression firstly,then used TSA,it enable to let p33ING1b re-expression significantly in colorectal cancer cell lines.Histone deacetylation and DNA hypermethylation coexisted in the process of p33ING1b inactivation,and hypermethylation was probably responsible for silence the p33ING1b.4.In the process of transcription inhibition of p33ING1b in colorectal cancer,except for methylation and acetylation of p33ING1b promoter,there maight have other mechanisms,such as other epigenetic modification mechanisms or abnormal changes of transcriptional regulatory mechanisms in its upstream.