The Mechanisms Of Migration And Invasion Of Bladder Cancer Though Regulation Of EMT By CCL18 Binding CCR8

Objective:Using Immunohistochemistry to detect the difference expression of CCL18 in bladder cancer and in paired normal tissues.Using Western blot technology to detect the difference expression of factors that are thought as the markers of EMT in different concentrations of CCL18 and inhibiting CCR8 by R243.Targeting the CCR8 gene in bladder cancer cells by shRNA interference technology,and then observed their biological behavior change.And discuss the specific molecular mechanism of CCR8 via CCL18 in migration,invasion and EMT of bladder cancer cells.Methods:1.Collection of BCa and noncancerous bladder tissues,Immunohistochemical analysis of CCL18 was conducted using 64 primary BC tissues and 30 adjacent noncancerous bladder tissues.To investigate the expression of CCL18 in different tissues and different grades of bladder cancer and its influence on tumor prognosis.2.Different concentrations of CCL18 were used to treat BCa cells(human bladder cancer 5637 cells),and CCR8 receptor inhibitor R243 was added to block the binding of CCR8 to CCL18.The expression of EMT related factors such as E-cadherin,MMP-2 and VEGF-C were compared by Western blot.3.Short hairpin(sh)RNA transfection.The human bladder cancer cell line(5637)was transfected with CCR8 shRNA and transfected non-target shRNA(NT shRNA)as the negative control group.The transfection efficiency of BCa cell line was observed by fluorescence microscopy,the expression of related genes was detected by qRT-PCR,the invasiveness of transfected tumor cells was detected by Transwell assay,and the expression of related proteins was detected by Western blot analysis.Result:1.The mRNA expression levels of CCL18 were significantly higher in BC tissues compared with in nontumor tissues.CCL18 mRNA expression was higher in BC tissues at stages III and IV compared with at stage II.Higher expression of CCL18 indicated higher tumor grade(P < 0.05).Kaplan-Meier analysis indicated that higher CCL18 expression was associated with worse patient survival(log?rank test,P=0.0487).2.The protein expression of EMT related factor E-cadherin decreased with the increase of ccl18 concentration(P < 0.05),while the protein expression of MMP-2 and VEGF-C increased with the increase of ccl18 concentration(P < 0.05).3.CCL18 enhances migration,invasion and EMT by binding CCR8 in bladder cancer cells.The expression of E-cadherin was up-regulated(P < 0.05),MMP-2,VEGF-C was down-regulated(P < 0.05),and the invasiveness of human bladder cancer 5637 cells was down regulated(P < 0.05).Conclusion:1.CCL18 can promote the migration and invasion of bladder cancer cells.2.The expression of CCL18 was correlated with the stage,the grading and prognosis of bladder cancer patients.The higher the expression,the worse the survival.3.CCL18 may promote tumor metastasis,invasion and EMT by binding with CCR8,a G protein-coupled receptor.