Relationship And Molecular Mechanism Between ATF3 And Colon Cancer Proliferation And Metastasis

BackgroundColorectal cancer(CRC)is one of the most common malignant tumors of the digestive tract in the world.In recent years,the incidence and death rate of colon cancer in China have significantly increased,which seriously threatens people’s health.Activated transcription factor 3(ATF3)is a member of the transcription factor family and is low in normal cells.However,signal stimulation can induce rapid expression and play a role in a short period of time.It is also known as an adaptive response gene.Studies have shown that ATF3 protein is involved in the development of many kinds of tumors,but its molecular mechanism of colon cancer cell line proliferation and migration is not unclear.ObjectivesTo investigate the relationship and molecular mechanism between ATF3 and colon cancer proliferation and metastasis,and to reveal the effect of overexpression/ downexpression of ATF3 expression on Wnt/β-catenin signaling pathway and EMT in colon cancer cell lines.Methods1.Immunohistochemical method was used to detect 49 cases of human colon cancer pathological tissue microarray(TMA)and analyze the expression of ATF3 protein in colon cancer tissues and its correlation with clinicopathological features.2.The basic expression level of ATF3 protein in colon cancer cell lines was detected by Western Blot,and cell lines with low expression levels were transfected into plasmids for overexpression experiments,and interference expression experiments were performed in highly expressed cell lines.3.The mRNA expression level of ATF3 in colon cancer cell lines was detected by qRT-PCR.4.The effect of ATF3 protein on proliferation of colon cancer cells was detected by CCK-8 assay.5.The effect of ATF3 protein on the migration of colon cancer cells was observed by cell scratching experiments,and the effect of ATF3 protein on cell migration was further verified by Transwell.6.The effect of ATF3 protein on the proliferation and migration of colon cancer cells was detected by Western blot.Results1.The high expression rates of ATF3 in colon cancer tissues and para-tumor lymph nodes were 42.9% and 10.0%,respectively,which was significantly lower than that of normal colon tissues 61.2%,P <0.01.2.The expression levels of ATF3 in HCT116 and HCT8 cells were 0.41±0.05 and 0.53±0.1,respectively.The expression levels of ATF3 in SW480 and RKO cells were 1.25±0.12 and 1.69±0.10,respectively,Shows that ATF3 has differential expression in colon cancer cell lines,F=116.64,P <0.01.3.The ATF3 over-expression plasmid was transfected into cells.The absorbance values of HCT116 cells and HCT8 cells for 48 h in the experimental and control groups were(1.47±0.10,1.77±0.12),(0.43±0.09,0.64±0.07);The ATF3 siRNA,RKO cells and SW480 cells measured the absorbance values of the experimental group and the control group at 48 h(1.52±0.21,0.96±0.14),(0.61±0.10,0.40±0.07);compared with the control group,there are significant differences,P <0.05.4.The ATF3 over-expression plasmid was transfected into cells.The numbers of transmembrane cells in HCT116 cells and HCT8 cells that counted in the experimental group and the control group for 48 hours were(35.0±15.2,79.3±15.9),(33.5±13.1,66.6±8.8),respectively.Comparing with each other,P<0.01;in the cells transfected with ATF3 siRNA,the number of transmembrane cells counted in RKO cells and SW480 cells at 48 h in the experimental group and the control group were:(114.8±10.2,79.6±9.4),(35.7 ± 7.2,15.1 ± 7.4),compared with the control group,there are significant differences,P <0.05.5.In the Wnt/β-catenin signaling pathway,ATF3 overexpression plasmids were transfected into HCT116 cells and HCT8 cells,and the expression levels of β-catenin,GSK-3β,and TCF7L2 proteins were significantly different from the control group,P<0.05 or P<0.01;siRNA transfected with ATF3 in RKO cells and SW480 cells,ATF3,β-catenin,GSK-3β,TCF7L2 protein expression,compared with the control group,there are significant differences,P <0.05 or P <0.01.In epithelial-mesenchymal transition(EMT)-related protein experiments,ATF3 overexpression plasmids were transfected into HCT116 cells and HCT8 cells,and the expression levels of E-cadherin and N-cadherin proteins;SW480 cells transfected with ATF3 siRNA,E-cadherin,N-cadherin protein expression,compared with the control group,there are significant differences,P <0.05.ConclusionATF3 can inhibits the proliferation and migration of colon cancer cells,it may inhibit the development of colon cancer by regulating the Wnt/β-catenin signaling pathway and EMT.