| Epigenetics is the study of inherited changes in phenotype or gene expression that do not alter DNA sequence.Recently,scientists have focused their attention on 5-hydroxymethylcytosine(5hmC)and a number of data has been accumulated regarding its structural formula,distribution,function of demethylation,or disease implications.Differences in 5hmC distributions may complicate previous observations of abnormal cytosine methylation statuses because of the incompetence of bisulfite sequencing PCR(BSP)to distinguish 5hmC from 5-methylcytosine(5mC).Therefore,new techniques are needed for the simultaneous detection of 5mC and 5hmC to identify new oncogene or tumor suppressor gene.In this study,we performed Methylation DNA immunoprecipitation sequencing(MedIP-seq)and Hydroxymethylation DNA immunoprecipitation sequencing(hMeDIP-seq)to synchronously measure these two modifications in six colorectal cancer samples.After identification of differentially methylated and hydroxymethylated genes in human colorectal cancer,we used High-throughput sequencing(RNA-seq)to identify whether the expression of these genes have relevance to their methylation or hydroxymethylation levels.So far,we found some candidates that are relevant to colorectal carcinogenesis.Then,we expanded the sample size to verify the mRNA expression of these candidates.We utilized theQuantiGenePlex assay to detect nearly 100 genes in 135 pairs of colorectal cancer tissues and the adjacent normal tissues and conducted data analyses to determine the prognostic value.As a result,we found both 5mC and 5hmC reduced in colorectal cancer.However,the local density of 5mC was increased in exon,CpG island and short interspersed repetitive sequence(SINE).But we were not able to see the same phenomenon from 5hmC,which had a great reduction in CpG shore,exon and region close to transcription start site(TSS).According to the content variation of 5mC,5hmC,we analyzed the differentially methylated region(DMR)and the differentially hydroxymethylated region(DhMR),which showed a semblable distribution in cancer tissues,compaired to the adjacent normal tissues.The DMR and DhMR concentrated in genebody,intron,intergenic region and repeat sequence.Methylation in DMRs was mainly up-regulated in CpG island,CpG shore and down-regulated in repeat sequence.DhMRs with increased 5hmC were in CpG dinucleotides of promoter,and decreased in CpG shore,enhancer and exon.We found the median expression of KIAA1199(0.112 vs.0.020)and SOX9(0.151 vs.0.083)was significantly increased in colorectal cancer tissues,while AQP8(0.005 vs.0.238).BEST4(0.004 vs.0.282)MAL(0.014 vs.0.194)and ADH1A(0.045 vs.0.713)expressed lower than the adjacent normal tissues(P<0.05;Friedman test).According to the mRNA expression,the Kruskal-Wallis test was applied to assess the association between all of these genes expression level and different clinicopathological variables.KIAA1199 mRNA level was increased in patients who were older than 60(0.13 vs.0.10;P=0.06).AQP8 expressed significantly higher in Duke's phase C&D than A&B(0.003 vs.0.008;P<0.05),BEST4(0.005 vs.0.003;P=0.06),MAL(0.012 vs.0.007;P<0.05)and ADH1A(0.071 vs.0.021;P<0.05)was associated with tumor size(small vs.large;P<0.05).There was no statistical difference between expression of these six genes and differentiation,histopathological type,tumor invasion,lymph node metastases,metastases,TNM stage(all P>0.05).KIAA1199(P=0.027),SOX9(P=0.043),AQP8(P=0.033),BEST4(P=0.004),MAL(P=0.045)and ADHIA(P=0.001)were associated with a poor outcome based on the Kaplan-Meier analysis.In conclusion,we found KIAA1199,AQP8,SOX9,BEST4,MAL and ADH1A,which were explored as oncogene candidates,having potential tumorigenesis functions and to become important molecular markers for predicting the progression and prognosis of colorectal cancer. |
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