The Expression And Mechanism Of EYA1 In Colorectal Cancer

BackgroundColorectal cancer is one of the most serious malignancies of the alimentary canal worldwide.According to the latest report of the world health organization(WHO),the incidence rate of CRC ranks the third among all malignancies and the fatality rate ranks the second.The EYA gene was first discovered by studying the eye development of fruit flies.EYA is a co-activator of SIX1,and the SIX-EYA complex was highly expressed in different types of tumors,including ovarian cancer,malignant perip Heral nerve sheath tumors,breast cancer and so on.The study found that expression level of SIX1 was abnormally up-regulated in human colorectal cancer,and the relationship between EYA1 and the occurrence,development and metastasis of colorectal cancer was not completely clear.ObjectivesThrough detecting the expression level of EYA1 in normal colonic mucosal epithelial cells NCM460 and colorectal cancer cell lines(HCT8,HCT116,LoVo,SW480 and RKO),the effect of EYA1 on the proliferation and migration of colorectal cancer cells was investigated by using LoVo cells that overexpressed EYA1 and siRNA interfered RKO cells.Methods1.The expression level of EYA1 mRNA in the TCGA(The Cancer Genome Atlas,TCGA)colorectal Cancer patients(n=337)was analyzed on Oncomine website(http://www.oncomine.org).The expression level of EYA1 in colorectal cancer tissues was detected by immunohistochemistry(IHC).The relationship between the expression level of EYA1 and survival in colorectal cancer patients was analyzed by the OncoLnc website(http://www.oncolnc.org).2.Western blot and real-time quantitative PCR(qRT-PCR)were used to detect the expression level of EYA1 protein and mRNA in normal colonic mucosal epithelial cells NCM460 and colorectal cancer cell lines(HCT8,HCT116,LoVo,SW480 and RKO).3.The overexpression vector of pc DNA3.1(+)-EYA1 was constructed and the low expression LoVo cells of EYA1 were transfected with Lipofectamine 2000.The siRNA synthesized by the company targeting EYA1 gene was transfected into RKO cells with high expression of EYA1,and the expression of EYA1 protein and mRNA after overexpression and interference was detected by Western blot and qRT-PCR.4.The effect of EYA1 on the proliferation of LoVo cells and RKO cells was analyzed by CCK-8 and clonal formation assay.The effect of EYA1 on the migration of LoVo cells and RKO cells was analyzed by scratch test and Transwell migration test.Results1.The expression level of EYA1 mRNA in TCGA(The Cancer Genome Atlas,TCGA)colorectal Cancer patients(n=337)was analyzed on Oncomine website(http://www.oncomine.org).The results showed that the expression level of EYA1 mRNA in colorectal cancer samples was significantly higher than that in normal colorectal tissues adjacent to cancer.The expression level of EYA1 in colorectal cancer tissues was detected by immunohistochemistry,and the results showed that the expression level of EYA1 in colorectal cancer tissue samples was higher than that in its relative normal colorectal tissues adjacent to cancer(P<0.05).The relationship between the expression of EYA1 and survival rate of colorectal cancer patients was analyzed through the OncoLnc website(http://www.oncolnc.org).The results showed that the expression level of EYA1 was negatively correlated with the survival period of colorectal cancer patients(P<0.05).2.The results of Western blot and qRT-PCR showed that EYA1 was relatively low in LoVo cells and high in RKO cells,so LoVo and RKO cells were selected for subsequent experiments.3.The results of Western blot and qRT-PCR showed that,compared with LoVo-Ctrl cells,the expression of protein and mRNA in LoVo-EYA1 cells were significantly up-regulated(P<0.05).However,compared with RKO-Ctrl cells,the expression of EYA1 protein and mRNA in RKO-si EYA1 cells were significantly down-regulated(P<0.05).4.The results of CCK-8 experiment showed that the growth rate of LoVo-EYA1 cells with transient overexpression of EYA1 was significantly higher than that of the control group(P<0.05).On the contrary,the growth rate of RKO-si EYA1 cells interfering with EYA1 was significantly lower than that of the control group(P<0.05).The analysis results of the clonal formation experiment showed that the number of clones formed in the LoVo-EYA1 group was significantly increased compared with that in the LoVo-Ctrl group(P<0.05).However,the number of clones in RKO-si EYA1 group was significantly lower than that in RKO-Ctrl group(P<0.05).5.The results of scratch experiment showed that the migration rate of LoVo-EYA1 cells overexpressed by EYA1 was significantly faster than that of the control group(P<0.05).On the contrary,the migration rate of RKO-si EYA1 cells after EYA1 interference was significantly slower than that of the control group(P<0.05).The results of transwell experiment showed that the migratory number of LoVo-EYA1 cells was significantly higher than that of the control group(P<0.05).On the contrary,the migratory number of RKO-si EYA1 cells was significantly lower than that of the control group(P<0.05).Conclusions1.The expression level of EYA1 in colorectal cancer tissues was significantly higher than that in the normal colorectal tissues adjacent to cancer.The expression level of EYA1 was negatively correlated with the survival of colorectal cancer patients.2.Overexpression of EYA1 promoted the migration and proliferation of colorectal cancer cells,while interference of EYA1 inhibited the migration and proliferation of colorectal cancer cells.