The Methylation Status And The Function Study Of CFTR In Colorectal Cancer

Objective:Colorectal cancer is a serious threat to human health,its morbidity and mortality are among the world’s top three malignant tumors.Relevant studies have found that abnormal DNA methylation leads to gene silencing and functional changes that are closely related to the formation of colorectal tumors,immune escape and invasion and metastasis.The research group used genome-wide methylation sequencing to screen the specific methylation of Cystic Fibrosis Transmembrane conductance Regulator(CFTR),which is associated with lymph node metastasis in colorectal cancer.The purpose of this study was to investigate the methylation level and function of CFTR in colorectal cancer.Patients and methods:(1)We performed genome-wide methylation sequencing to screen genes that exhibited fold change Log2FC>|1| and P<0.05 in 12 colorectal cancer(CRC)tissues,metastatic lymph nodes and adjacent normal tissues which were preserved by the pathology department of Qianfoshan Hospital affiliated to Shandong University.(2)Methylation specific PCR(MSP)was used to detect CFTR promoter methylation status in 70 CRC samples(including 12 samples for sequencing).Meanwhile,the relationship between methylation status of CFTR promoter in CRC tissues and clinicopathological parameters was analysed.(3)Immunohistochemistry(IHC)was used to detect the expression of CFTR protein in normal colorectal tissues and corresponding cancer tissues.(4)The correlation between the methylation state of CFTR promoter and its mRNA expression level was investigated by methylation-specific PCR(MSP)and quantitative Real-time Polymerase Chain Reaction(qRT-PCR).(5)Colony formation,transwell,and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to evaluate the effect of CFTR overexpression in CRC cells.Results:(1)The results of genome-wide methylation sequencing showed that the methylation level of CFTR in primary and lymph node metastatic cancer tissues was higher than that in normal intestinal mucosal tissues,P=0.004,Log2FC=4.41.(2)MSP validation results showed that the methylation frequency of CFTR promoter in 70 colorectal cancer tissues was 62.2%(45/70),which was higher than 41.4%(29/70)of normal intestinal mucosal tissues,and the difference was statistically significant.(x2=7.338,P<0.01).Combined with clinical data analysis,the methylation of CFTR promoter was significantly associated with age,degrees of differentiation and lymph node metastasis in CRC tissues(P<0.05).(3)The positive expression rates of CFTR protein in normal intestinal mucosa and colorectal cancer tissues were 62.9%(44/70)and 45.7%(32/70),respectively,with statistically significant differences(Χ2=4.145,P<0.05).(4)The methylation of CFTR was negatively correlated with its mRNA expression in colorectal cancer(Χ2=4.227,P<0.05).(5)Results of transwell,MTT,and colony formation assays showed that CFTR overexpression suppressed the migration,invasion and proliferation of CRC cells.Conclusion:(1)CFTR is frequently methylated in human colorectal cancer tissues,and CFTR methylation is associated with lymph node metastasis,differentiation degree and age of patients with colorectal cancer.(2)The expression of CFTR may be regulated by promoter methylation in colorectal cancer tissues and cells.(3)Overexpression of CFTR can inhibit the proliferation,migration and invasion of colorectal cancer cell lines,suggesting that CFTR plays a role as an anticancer gene in human colorectal cancer.