The Mechanism Study Of YWHAE Regulates EMT Of Colon Cancer Cells By AKT Signal

Background: IARC,the International Agency for Research on Cancer,the World Health Organization’s International Agency for Cancer Research,released the latest global cancer burden data for 2020,colorectal cancer(CRC)incidence in China and the world ranked second and third,respectively;mortality ranked fifth and second,respectively,invasion,metastasis,recurrence are common causes of death.Previous studies have shown that,Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein(YWHAE)that highly expressed in multiple tumors,including CRC,involved in regulating a variety of cell functions,such as cell cycle,apoptosis,diffusion migration,through binding to proteins that associated with signal transduction mediated by serine phosphate.High expression of YWHAE was significantly associated with higher risk of CRC metastasis and lower patient survival.However,the molecular mechanism of YWHAE how to regulate CRC transfer is unclear.Objective: To investigate the correlation between the expression of YWHAE and epithelial-mesenchymal transition(EMT)of colon cancer and its possible molecular pathways.Methods: To construct YWHAE short hairpin RNA that was packaged by lentivirus(LV-YWHAE-shRNA)and infect the SW620 cells of colon cancer.The experiment was divided into YWHAE interference group(KD group),negative transfection control group(NC group)and non-transfection group(CON group).Using Western blot,RT-q PCR and immunohistochemistry(IHC)to detect the expression of YWHAE genes in colon cancer cells before and after infection;Cell cloning test compared the ability of colon cancer cell cloning before and after infection;MTT method detected the proliferation ability of SW620 cells of colon cancer before and after infection;Using Western blot and IHC to detect the markers of EMT and pathway proteins that was related to AKT-β-catenin,ERK1/2,to analyze the possible signaling pathways that involved regulating EMT of colon cancer cells by YWHAE genes.Results: LV-YWHAE-shRNA successfully infects colon cancer SW620 cells,the infection rate is more than 90%;The results of q PCR and Western blot showed that the inhibition efficiency of SW620 cells in KD group was 75%,compared with NC group(P<0.05);There was no significant difference in expression between the NC group and the CON group(P>0.05).MTT experimental results showed that the growth multiples of 96 h were 1.51,3.04,3.11,respectively,The cell growth rate in KD group was 50.3% lower than NC group,Differences were statistically significant(P<0.01);Compared to CON group,There was no significant difference between the NC group(P>0.05).The cell clone formation test showed that the number of cell clones in CON group,NC group and KD group were 1039,1131,191,respectively.Compared with NC group,the number of cell clones in KD group decreased by 83.1%,the difference was statistically significant(P<0.05);The area of cell clones in CON group,NC group and KD group were 94.37,93.58,74.19,respectively.Compared with the NC group,the area of cell clones in KD group decreased by 20.7%,the difference was statistically significant(P<0.05);There was no significant difference between NC group and CON group(P>0.05).the results of Western blot show that,Compared to NC group,the expression of E-cadherin in KD group was upregulated by 41.1%,difference was statistically significant(P<0.05);Compared to NC group,the expression of Vimentin in KD group was decreased by 68.8%,difference was statistically significant(P<0.05);There was no significant difference between NC group and CON group(P>0.05).the results of IHC showed that the positive expression rates of E-cadherin were 81.2%,30.6%,50.8% in the KD,NC and CON groups.Compared to cells in NC group,The expression rate of E-cadherin’s positive cell in KD group up-regulated by 62.3%,differences were statistically significant(P<0.05);the positive expression rates of Vimentin were 1.4%,71.2%,70.4% in the KD,NC and CON groups.Compared to NC group,the expression rate of Vimentin’s positive cell in KD group decreased by 98.0%,differences were statistically significant(P<0.05).Compared to CON group,There was no significant difference in the expression of E-cadherin,Vimentin in NC group((P>0.05).The results of western blot show that,Compared to NC group,the expression of AKT,p AKT protein in KD group downregulated by 75.0%,77.8%,their differences were statistically significant(P<0.05).Compared to CON group,There was no significant difference in the expression of AKT,p AKT,β-catenin expression in NC group(P>0.05);the expression of ERK,p ERK protein was no significant difference in KD group,NC group and CON group(P>0.05);the results of IHC show that,the positive expression rates of AKT were 30.2%、94.2%、61.5% in KD,NC,CON groups.Compared to NC group,the expression of AKT in the KD group decreased by 67.9%,differences were statistically significant(P<0.05);The positive expression rates of p AKT were 60.4%、95.5% 、 96.1%in KD,NC,CON groups.Compared to NC group,The positive expression rate of p AKT in KD group decreased by 36.8%,differences were statistically significant(P<0.05);The positive expression rates of β-catenin were20.3%、90.6%、84.7% in KD,NC,CON groups.Compared to NC group,The positive expression rate of β-catenin in KD group decreased by 77.6%,differences were statistically significant(P<0.05).Compared to CON group,the expression of AKT,p AKT,β-catenin were no significant difference in NC group(P>0.05);the expression of ERK,p ERK were no significant difference in KD,NC and CON group(P>0.05).Conclusion: Silencing YWHAE can inhibit the growth and cloning of colon cancer cells,and block the occurrence of EMT,which may be related to block the signaling pathway of AKT.