Study Of Relationship Between CHD5Methylation And Colorectal Carcinogenesis And Clinical Pathological Features

Objective: Colorectal cancer is ranked third both in the incidence andmortality rate of malignant tumor diseases in the world at present which isranked three and fifth in the incidence and mortality respectively in China. Ithas become a serious threat to human health. However, its pathogenesis is notclear. In recent years, TSGs’methylation has become the hotspot of colorectalcancer study. Aberrant DNA methylation can result in tumor suppressor geneinactivation and cannot transcribe or translate normally, then will lose theanticancer effects. Then cell monoclonal proliferation happens and tumorforms. So it participates in the genesis and progression of colorectal cancer.Many studies suggest that CHD5(chromodomain helicase DNA bindingprotein5) is a tumor suppressor gene of colorectal carcinoma，which has thefunction of regulating cell proliferation, apoptosis and senescence as the totalswitch of tumor suppression system. In recent years,foreign scholars havefound that down-regulation or silencing of CHD5caused by abnormalmethylation is associated with a variety malignant tumor，including colorectalcancer. The domestic scholars only find the decrease or loss of CHD5expression is related with colorectal cancer.But the relation between CHD5methylation and colorectal cancer has not been reported. This paper aims tostudy the relationship between CHD5methylation and development，as wellas the clinicopathological factors of colorectal cancer.Methods: A total of40patients with colorectal cancer at the SecondHospital and the Fouth Hospital of Hebei Medical University and40patientswith adenoma at the Second Hospital from March to August2013wereenrolled. Colorectal cancer tissues, paracancerous tissues, normal tissues andadenoma tissues specimens were collected. The paracancerous tissues were collected at least more than5cm from the cancer tissue and the normal onesfrom operation margin tissues. All the specimens were confirmed by pathology.In this paper MSP (methylating-specific PCR) technique was used to analyseCHD5methylation status in the specimens above,as well as the correlationbetween CHD5methylation and clinicopathological factors in CRC such asage, gender, tumor size, tumor grade and stage. We also analyzed thecorrelation between CHD5methylation and clinicopathological factors ofadenoma specimens such as age,gender, adenoma size, number andpathological type. Reverse transcription real time polymerase chainreaction(RT-PCR)was performed to detect the mRNA expression of CHD5inthe four kinds of tissues.Western-blot was performed to detect the expressionof CHD5protein of tissues above.We at last analyzed the correlation betweenthe expression of CHD5mRNA and protein in CRC tissues,as well as thecorrelation between CHD5methylation and the expression of CHD5protein.Results:1Methylation of CHD5gene in tissues1.1Hypermethylated CHD5was found in12.5%（5/40）、22.5%（9/40）、47.5%（19/40）、72.5%（33/40）of samples from normal mucosa tissues, paraca-ncerous tissues, adenoma tissues, and cancer tissues, respectively. CHD5met-hylation rate in cancer tissues was significantly higher than normal mucosatissues, paracancerous tissues and adenoma tissues (X2=29.463, X2=20.050,X2=5.208,P<0.05). CHD5methylation rate in adenoma tissues was significantlyhigher than normal mucosa tissues and paracancerous tissues(X2=11.667, X2=5.495,P<0.05). However the differences between normal and paracanceroustissues have no sense in statistics(X2=1.385,P>0.05).1.2CHD5methylation of colorectal cancer tissues has positive relation withdifferentiation,tumor stage,lymph node metastasis and distant metastasis(X2=4.742,X2=4.409,X2=5.230,X2=7.905,P<0.05).But no significant associati-ons were observed between hypermethylated CHD5and clinicopathologicalfeatures including sex, age, tumor location, tumor size, and pathologicaltype(P>0.05). 1.3CHD5methylation of adenoma tissues has positive relation withpathological type(X2=9.256,P<0.05). CHD5methylation rate of villousadenomas（4/12,75.0%）is obviously higher than tubular adenomas（12/16,33.3%），tubulovillous adenomas（3/9,33.3%）， and serrated ones（0/3,0%).But no significant associations were observed between hypermethylatedCHD5and clinicopathological features of adenoma tissues including age,gender, adenoma location,size and number (P>0.05).2Expression of CHD5mRNA in four kinds of tissuesThe expression of CHD5mRNA was0.225±0.276,0.169±0.231,0.147±0.159,0.013±0.011in normal tissues, paracancerous tissues, adenoma,and cancer tissues. Analyzed with one-way anova SNK method, differenceexisted in expression of CHD5mRNA of normal, paracancerous, adenomaand cancer tissues(P<0.05),presenting a declining trend.3Expression of CHD5protein in four kinds of tissuesThe expression of CHD5protein was0.438±0.205,0.398±0.180,0.156±0.100,0.024±0.311. Analyzed with one-way anova SNK method, differenceexisted in expression of CHD5protein of normal, adenoma and cancertissues(P<0.05), presenting a declining trend.4Correlation between CHD5mRNA and protein in cancer tissuesUnder the Pearson correlation analysis, the expression between CHD5mRNA and protein has the positive correlation(r=0.899,P<0.05). The decreaseor deletion of CHD5mRNA is always associated with the decrease or missingof CHD5protein expression.5Correlation between CHD5methylation and protein expression in cancertissuesWe detected the CHD5methylation and CHD5protein expression in40cancer tissues qualitatively. In the23cases which CHD5protein expressionwas lost, the CHD5methylation rate was87.0%(20/23). While in the17caseswhich CHD5protein expression was positive,the CHD5methylation rate was52.9%(9/17).Significant differences were observed between the methylationrate (X2=5.673,P<0.05). That is, methylation rate of cases which CHD5 protein expression was negative is significantly higher than protein expressionof positive ones.Conclusion:1CHD5gene methylation rate gradually increases in the evolutionprocess of colorectal cancer.CHD5methylation may be related with thegenesis and progression of colorectal cancer; CHD5methylation rate inadenoma is higher than normal and paracancerous tissues while cancer tissuesis significantly higher than adenoma tissues.It suggests CHD5methylationmay participate in the colorectal adenoma-carcinoma evolution process.2The high CHD5methylation rate in colorectal cancer is accompaniedwith the decrease expression of mRNA and protein. The expression betweenCHD5mRNA and protein has the positive correlation.The CHD5methylationis correlated with loss expression of CHD5protein. The experiment suggestsCHD5methylation may cause silence of expression, then lead to genesis andprogression of colorectal cancer.3CHD5methylation is obvious associated with the clinical andpathological features of colorectal cancer. CHD5methylation is higher in CRCwith poor differentiation, lymph node metastasis, distant metastasis and badtumor stage.