The Study On The Clinical Significance Of NDRG4 Gene Methylation And The Influence Of NDRG4 To The Biological Behavior In Colorectal Cancer

PurposeTo explore the relationship between the methylation of the promoter region of NMyc downstream regulated gene 4(NDRG4)and the clinicopathological features of colorectal cancer,and to analyze its potential diagnostic value and clinical significance in colorectal cancer.Gene expression profiling interactive analysis(GEPIA)database was used to analyze the NDRG4 expression level in colorectal cancer and normal tissues.Then we constructed colorectal cancer cell line with stable overexpression of NDRG4 and investigated the mechanism of NDRG4 gene influencing the biological behavior of colorectal cancer cell line.Methods(1)The samples of colorectal cancer(n=43)and paired normal tissues(n=24),were collected from January 2014 to December 2016.The methylation rate and methylation degree in the promoter of NDRG4 gene in each sample were analyzed by quantitative methylation specific PCR(q MSP).The relationship between the positive rate of NDRG4 methylation and clinicopathological features in colorectal cancer.The diagnostic value of NDRG4 promoter methylation in colorectal cancer was evaluated by sensitivity,specificity and receiver operating characteristic curves(ROC).GEPIA database was used to analyze the NDRG4 expression level in colorectal cancer and normal tissues.(2)We constructed a colorectal cancer cell line HCT116 with stable expression of NDRG4,and used q RT-PCR and western blotting to verify whether the stable cell line was successfully constructed.CCK-8 assay was used to detect the changes of cell growth and survival before and after overexpression.Clone formation assay was used to detect the changes of cell population dependence and proliferative ability.Transwell assay was used to detect the changes of cell migration and invasion abilities.Flow cytometry was used to detect the changes of cell apoptosis and cell cycle.The effect of NDRG4 overexpression on p-AKT(Ser473)expression was detected by western blotting.ResultsThe positive rates of NDRG4 promoter methylation in colorectal cancer and paired normal tissues were 65.12%(28/43)and 4.17%(1/24),respectively.Compared with paired normal tissues,NDRG4 promoter methylation was more highly methylated and more easily detected in colorectal cancer tissues(P=0.000,P=0.000).The area under curve(AUC)to distinguish colorectal cancer and paired normal tissues was 0.8232(P=0.000),the sensitivity and specificity were 65.12% and 95.83%,respectively.The detection rate of NDRG4 promoter methylation was not associated with clinicopathological features of colorectal cancer(P>0.05).The NDRG4 gene expression level in normal tissues was significantly higher than colorectal cancer(P<0.01).Overexpression of NDRG4 significantly inhibited the growth activity,cell clone formation ability,cell migration and invasion ability,and induced cell apoptosis,reduced S phase cells.Overexpression of NDRG4 significantly reduced the expression of p-AKT(Ser473)in colorectal cancer cell.ConclusionNDRG4 promoter methylation is a potential effective molecular marker for screening colorectal cancer.Overexpression of NDRG4 can significantly inhibit the growth,proliferation,apoptosis,migration and invasion of colorectal cancer cells.NDRG4 gene can play a role of tumor suppressor gene in colorectal cancer cells by inhibiting PI3K/AKT signaling pathway.