Methylated CLDN11 Associated With The Metastasis Of Colorectal Cancer

ObjectiveColorectal cancer(CRC)is one of the most common malignancies of gastrointestinal neoplasms,involving the malignant transformation of normal intestinal epithelial cells resulting from the accumulation of abnormal genetic and epigenetic changes.At present,the 5-year survival rate of patients with advanced CRC is only 30%,and the problem of low survival rate needs to be solved urgently.Metastasis of CRC is the main reason for the low survival rate of CRC.Therefore,it is critical to understend the mechanism of CRC metastasis.DNA methylation is one of important component of the epigenetic modification,which has been proven associatied with the development of CRC intriguing numerous attentions.A large number of literature reported that epigenetic modification occurred in early stage of CRC,even earlier than genetic changes.DNA methylation has a great potential in early diagnosis of CRC.In the current study,we attempted to analyze that whether the abnormal methylated genes occurred in the early stage of CRC has a potential in prediction of CRC metastasis and whether the methylation change of these methylated genes could affect the migration ability of CRC cells,which can not only help to understand the pathogenesis of CRC metastasis and monitor the development of CRC in real time,but also provide a theoretical basis for the new strategy of colorectal cancer treatment.Therefore,this study aimed to analyze the abnormal process of DNA methylation during CRC,and analyze its relationship with CRC metastasis.Methods1.Differential methylated genes were identified between colorectal cancer and corresponding normal tissues using the Illumina humanmethylaiton 450 K chip(HM450K);Gene functional annotation of differentially methylated genes was analyzed by DAVID(https://david.ncifcrf.gov/).Based on the results of HM450 K,we found that CLDN11 involving into cellular adhesion molecules(CAMs)pathway was hypermethylated in CRC.Therefore,we attempt to analyze the role of methylated CLDN11 played in CRC;2.Then,we examined the methylation level of CLDN11 of 125 CRCs and corresponding normal tissues using quantitative methylation specific polymerase chain reaction(qMSP).The relationship of the methylation of CLDN11 and clinicalpathological characteristics of CRC were also analyzed;3.Luciferase reporter assay was performed to assess the transcriptional ability of the fragment of CLDN11 tested in qMSP;4.The expression of CLDN11 mRNA in CRC tissues and corresponding normal tissues were detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).The correlation of CLDN11 methylation and expression was analyzed;5.The CLDN11 methylation and expression level of four CRC cell lines(HCT116,COLO205,SW620 and HT29)and normal intestinal epithelial cell line(NCM460)were analyzed by qMSP and qRT-PCR;6.The methylation status of CLDN11 was converted by demethylation agent(5-aza-dC),and the methylation and expression of CLDN11 was detected by qMSP and qRT-PCR to confirm optimized concentration of 5-aza-dC;7.Transwell migration experiment was performed to observe the migration ability change of CRC cell lines after reversing the methylation level of CLDN11;8.The reverse correlation of CLDN11 methylation and expression,as well as the associateon between CLDN11 methylation and CRC metastasis,were justified by the methylation,expression and clinical data from TCGA.Results1.According to the results of HM450 K assay,4047 differentially methylated CpG sites were identified,covering 1725 reference genes.Of these,1017(25%)sites were hypermethylation CpG sites.After all the differentially methylated CpG sites were annotated,we found the hypermethylation of CLDN11 of CAMs pathway in CRC tissues;2.The hypermethylation of CLDN11 in CRC tissues was observed when compared with normal tissues(P = 3.71E-23).Then,we analyzed the association of CLDN11 methylation and clinicapathological parameters.And the result showed that the methylation level of CLDN11 was higher in CRCs with lymph node metastasis than in CRCs without(P = 0.01);3.The luciferase reporter gene assay showed that the fragment of CLDN11 promoter tested in qMSP could regulate the expression of downstream gene(P = 0.002);4.The results of qRT-PCR showed that the expression level of CLDN11 mRNA was decreased in CRC tissues(P = 0.002).The reverse correlation of CLDN11 methylation and expression was found,suggesting that promoter hypermethylation of CLDN11 is responsible for its downregulation(r =-0.19,P = 0.04);5.Among the four CRC cell lines,the hypermethylation of CLDN11 were found in HCT116,COLO205 and HT29.While,the methylation of CLDN11 in SW620 was lower than that in NCM460.As to the expression of CLDN11,the lower expression of CLDN11 was observed in HCT116,COLO205 and HT29 than in NCM460,and the expression of CLDN11 in SW620 was remarkable higher than in NCM460.These results supported that hypermethylation of CLDN11 reduced the expression of CLDN11 mRNA.6.According to the methylation and expression level of CLDN11,we determined to treat HCT116 cell line with different concerntrations of demethylation agent(5-aza-dC).Then,the methylation and expression level of CLDN11 were detected for the identification of the optimized concerntation of 5-aza-dC.The results showed that the methylation of CLDN11 was significantly changed when HCT116 cell lines was treated with 9 ?mol/L 5-aza-dC.7.The Transwell assay showed the debased migration capacity of HCT116 cell line after treated with 9 ?mol/L 5-aza-dC;8.Based on TCGA methylation and expression data,the results confirmed the reverse correlation of CLDN11 methylation and its expression(r =-0.21,P = 0.000035).Besides,the hypermethylation of CLDN11 is associated with low relaspe free survival(RFS)in CRC(Log rank P = 0.04).ConclusionsOur results show that CLDN11 methylation is involved in CRC metastasis.Analysis of large sample data from public database shows that CLDN11 is a potential molecular marker for predicting relaspe free survival(RFS)in CRC.