The Effect Of WNT5A Promoter Region Methylation On Chemotherapy And Prognosis Of Colorectal Cancer Patients

BackgroundThe incidence of colorectal cancer in China has been increasing in recent years.In developed cities and regions,the incidence of colorectal cancer has risen to the third place,and to the fifth place in rural areas for malignant tumors.Globally,the incidence of colorectal cancer has also increased year by year,and the incidence of colorectal cancer has ranked third in the world for malignant tumors.The incidence of colorectal cancer is the fourth highest in the United States,and the fatality rate is the second in the United States.WNT signaling pathway plays an important role in embryonic development and tumorigenesis.WNT signaling pathway is often found to be dysregulated in multiple types of cancers.It is particularly important in colorectal cancer(CRC),as the majority of the CRC cases are characterized with mutations in the adenomatous polyposis coli(APC)gene,a negative regulator of WNT signaling,resulting in aberrant activation of WNT canonical signaling.Aberrant activation of signal transduction pathway and accumulation of ?-catenin protein in the nucleus,Inhibiting the activity of WNT signaling pathway can inhibit the mutation of APC gene in colon cancer.The mutation of APC gene was found in colorectal cancer more than 85%,which block the phosphorylation of ?-catenin and induce ?-catenin,intracellular aggregation of tenin activates Wnt /?-catenin pathway and induces colorectal cancer.WNT5A as an important member of the WNT family,is located in the 3p14.2-p21.1 region of chromosome,which contains 5 exons.The promoter region is located in a region which rich glycerophosphatidylcholine and contains multiple cis-acting elements.The 631 base pairs at the start of WNT5 A contain strong promoter activity.The gene encodes a growth factor which rich in cysteine and play a important on intercellular signaling is involved in the process of growth and differentiation.WNT5A is an important ligand in the noncanonical WNT pathway,include two protein subtypes: WNT5A-long(inhibit tumor proliferation)and WNT5A-short(promote tumor proliferation).WNT5 A produces different protein subtypes and the different pathway mediated by it which produce a diametrically opposite effection: in different tumor and different stages of tumor.WNT5 A,as an inhibitor of WNT /?-catenin signaling pathway,is usually detected hypermethylation in colorectal cancer.Based on the above background,we studied the prognostic value of WNT5 A methylation and its potential molecular mechanism in cultured CRC cells.In this study,HCT116,HT29,SW620,HCT8,LoVoSW480 and Rko colon cancer cell lines were studied.WNT5 A mRNA level was detected by qRT-PCR.Determination of DNA methylation by MSP.The effect of 5-FU on the viability of colorectal cancer cells was determined by MTT assay.To further investigate whether WNT5 A methylation is related to drug reaction and progressive survival in patients with colon cancer treated with 5-fluorouracil chemotherapy.Method1.Detection of mRNA level of WNT5 A in 7 Colorectal Cancer cells by qRT-PCR.Determination of DNA methylation by MSP.DAC treatment of HCT116 and SW620 cells with low expression of WNT5 A,then detection of WNT5 A expression by qRT-PCR.2.HCT116 and SW620 cells with low expression of WNT5 A were transfected with WNT5 A plasmid to make WNT5 A overexpression.Lovo,SW460 cells with high expression of WNT5 A were transfected with WNT5A-siRNA to make WNT5 A low expression.Then processing with 5-FU and detect the cell viability by MTT.3.Collect 126 cases of colorectal cancer from 2000 to 2001 in the first affiliated Hospital of Zhengzhou University,analysis of the relationship between WNT5 A methylation and clinicopathological characteristics and the effect of 5-FU chemotherapy and survival time without progression.4.Statistical analyses were conducted using GraphPad Prism 6(GraphPad Software,Inc.).Data were presented as mean±SEM.Statistical significance was determined by Student'st-test for pairwise comparisons or by one-way ANOVA with Tukey's multiple comparison tests or two-way ANOVA with Bonferroni's multiple comparison tests for multiple comparisons.Survival curve was created using log-rank(Mantel–Cox)test.p < 0.05 was considered statistically significant.Result1.The methylation level of WNT5 A promoter region affects its transcription level.The transcriptional level of WNT5 A and its promoter methylation were detected in several colorectal cancer cell lines.It was found that the promoter region of WNT5 A was low or non-methylated in LoVoSW480 and Rko cells,and its mRNA level was higher.In HCT116,HT29,SW620 and HCT8 cells,the WNT5 A promoter region was completely or hypermethylated which mRNA level was low.WNT5 A promoter hypermethylated colorectal cancer cell lines HCT116 and SW620 were treated with DAC to inhibit WNT5 A promoter methylation,it was found that WNT5 A transcriptional level was up-regulated.These results indicated that methylation in the promoter region of WNT5 A can affect its transcriptional level.2.Overexpression of WNT5 A in HCT116 and SW620 cells with WNT5 A promoter region hypomethylation which abolished the tumor inhibition of 5-FU.Knockdown of WNT5 A in LoVo and SW480 cells with WNT5 A promoter region low methylation which enhanced the tumor inhibition of 5-FU.3.There is no correlation between WNT5 A promoter region methylation and tumor stage,histological grade,sex and age in colorectal cancer patients.The methylation level of WNT5 A promoter region in colorectal cancer patients who were treated by 5-FU was significantly higher than those colorectal cancer patients who were not treated with 5-FU.Colorectal cancer patients with hypermethylation of the WNT5 A promoter had longer progression-free survival than those with low methylation.CONCLUSION1.The expression of WNT5 A in colorectal cancer is related to the methylation level of its promoter regions.2.WNT5 A can affect the sensitivity of colorectal cancer cells to 5-FU and the tumor inhibition of 5-FU to the colorectal cancer.3.WNT5 A promoter region Methylation is associated with the effect of 5-FU chemotherapy and prognosis of colorectal cancer patients.Therefore,WNT5 A could be used as an biomarker to judge the prognosis of colorectal cancer and select effective chemotherapy.