Claudin-6 Inhibits The Proliferation,migration And Invasion Of Prostate Cancer Cells

Prostate cancer is a prostate-derived epithelial malignancy.In the late stage of prostate cancer,the metastasis and recurrence of tumor cells are the main cause of death in patients.The current study suggest that the process of the growth and metastasis of tumor are regulated by multiple factors,among which tight junction(TJ)is closely related to the proliferation,movement,invasion,and other metastatic phenotypes of tumor cells.The tight junction proteins CLDNs are the main components of tight junctions(TJs).Their abnormal expression can affect the tight junctions' structure,function and the related cell signaling,which play an important role in the occurence and metastasis of epithelial-derived malignant tumors.Claudin-6,one of CLDNs family of tight junction proteins,acts a pivotal part in maintaining the tight junction structure and function between cells.In our previous study,we found that claudin-6 significantly inhibited the migration and invasion of breast cancer,cervical cancer,and colon cancer cells.We speculated that the silence of claudin-6 expression may play a role in the metastasis of epithelial-derived malignant tumors.At present,it has not been reported whether claudin-6 can inhibit prostate cancer cell invasion phenotype.It's the first study that using molecular techniques to explore the effect of claudin-6 on proliferation,migration and invasion of prostate cancer,which will provide some theoretical support for the treatment and control of prostate cancer.Purpose:To investigate the effect of overexpression of CLDN6 on the biological phenotype of prostate cancer cells.Methods:The expression of claudin-6 gene and protein in human prostate cancer cells LNCAP and DU145 was detected by RT-PCR and Western Blot.The prostate cancer cells LNCAP and DU145 were transfected by latent virus carrying claudin-6.Puromycin was used to screen for stable clone with overexpression of claudin-6;RTPCR,Western Blot and cell immunofluorescence were applied to identify the transfection efficiency of lentiviral and the expression and localization of claudin-6 protein in the stable cell clone.The cell viability test CCK8 assay was used to detect the proliferation of prostate cancer cells and the cell growth curve was drawn;flow cytometry was used to test the distribution of prostate cancer cell cycle after the overexpression of claudin-6;the resistance experiment was used to detect the monolayer transepithelial resistance of prostate cancer cells;the inverted microscope was used the to observe the morphological changes of prostate cancer cells after the overexpression of claudin-6;phalloidin fluorescence staining was used to observe the skeletal changes of prostate cancer cells after the overexpression of claudin-6;The cell scratch test and Transwell chamber assay was used to detect cell migration and invasiveness;Western Blot was used to detect the expression of EMT related proteins after the overexpression of claudin-6.Results:RT-PCR and Western Blot showed low expression of CLDN6 in prostate cancer cells LNCAP and DU145.LNCAP and DU145 clones overexpressing CLDN6 were obtained after transfection of lentivirus.RT-PCR and Western Blot showed the expression levels of RNA and protein of CLDN6 in the clone group were significantly higher than those in empty group.The results of immunofluorescence staining showed that CLDN6 was expressed in the clone group and was mainly expressed on the cell membrane,but no obvious expression was observed in the empty group.The results of CCK-8 cell activity showed that compared with the empty group,the proliferation ability of the cells in the clone group decreased.The results of the cell cycle assay showed that the cell proliferation ability of the clone group was lower than that of the empty group.The transepithelial resistance experiment showed that the monolayer transcellular epithelial resistance between the cells in the clone group was significantly higher than that in the empty group.The morphology of prostate cancer cell after the overexpression of claudin-6 was observed using inverted microscope,founding that the transition to epithelial cell morphology,showing as polygonal,flat cell body and unclear boundary.The results of phalloidin fluorescence staining showed that the fluorescence intensity of F-actin in the prostate cancer cells was reduced after the overexpression of claudin-6,and the microfilament structure in the cytoplasm was not obvious.The cell scratch test and the Transwell cell migration experiment showed that the migration ability of the clone group was significantly lower than that of the empty group.Transwell cell invasion assay results showed that the invasive ability of the clone group was significantly lower than that of the empty group.Western blot showed that after the overexpression of claudin-6,the expression of epithelial cell marker E-cadherin was significantly up-regulated,while the interstitial cell markers N-cadherin and Vimentin were significantly downregulated.Conclusion:The overexpression of claudin-6 can inhibit the proliferation,migration,invasion and reverse the EMT process and enhance the tight junction of prostate cancer cells.