Effects Of CLDN6 On Proliferation,migration And Invasion Of Hepatoma Carcinoma Cells And Its Expression In Liver Cancer Tissue

Primary hepatocellular carcinoma,or hepatocellular carcinoma,is one of the most highly pathogenic malignant tumor in China.Metastasis and recurrence are the main causes of death in patients with liver cancer.Study found that in a variety of epithelial malignant tumors,the claudins(CLNDs)abnormal expression can influence tight junctions(TJs)function and then plays an important role in the occurrence,development and metastasis of malignant tumor.The TJs consists of the junctional adhesion molecule(JAMs),zonula occludens(ZOs),occludin and claudin(CLDNs),As the skeleton protein of TJS,CLDNs' function is the most important.CLDN6 is one of the important members of the family of CLDNs,CLDN6 genes were identified in the previous experiments,it is suppressor genes for breast cancer,and found CLDN6 high expression in human hepatocellular carcinoma tissue,but the expression of CLDN6 in hepatoma carcinoma cells and its effect on the biological behavior of hepatoma carcinoma cells are not clear.Therefore,this study will explore the effects of CLDN6 on proliferation,migration and invasion of HepG2 and Hep3 B and their clinical significance in liver cancer tissues.Methods:We studied the expression and localization of CLDN6 in human hepatoma carcinoma cells lines by reverse transcriptase-PCR(RT-PCR),western blot and cellular immunohistochemistry.The CLDN6 gene in HepG2 and Hep3 B was silenced by liposomal transfection method,and the silent CLDN6 gene of human hepatoma carcinoma cell line was obtained by G418 stable screening.The silencing efficiency of CLDN6 in HepG2 and Hep3 B was identified by RT-PCR,western blot and cellular immunofluorescence assay.The proliferation of cells was detected by CCK8 and flow cytometry.Wound-healing assay and transwell chamber method were used to detect the migration of cells.Transwell chamber method was used to detect the invasion ability of cells.Western blot was used to detect the expression of EMT related proteins in human hepatoma carcinoma cells.Finally,we used immunohistochemical staining techniques to detect the expression of CLDN6 in human liver cancer tissues and to analyze the relationship between them and clinicopathological parameters of liver cancer.Results:1.Expression of CLDN6 in human hepatoma carcinoma cells.RT-PCR and western blot results showed that the mRNA and protein expression levels of CLDN6 in HepG2 and Hep3 B were significantly higher than those of normal liver cells,and the difference was significant(P<0.05).Cytoimmunological experiments also showed that CLDN6 protein in HepG2 and Hep3 B was strongly positive and expressed mainly in cell membrane.2.Silence and identification of CLDN6 gene in human hepatoma carcinoma cells.Using liposome transfection method in silence CLDN6 genes of HepG2 and Hep3 B,and stable by G418 screening,get a silent CLDN6 monoclonal cell line HepG2-sh and Hep3B-sh,by fluorescence microscope to observe the transfection efficiency.We used RT-PCR to detect the mRNA expression levels of CLDN6 in 1-12 clones of HepG2-sh and 1-8 clones of Hep3B-sh,the results found CLDN6 mRNA expression level significantly decreased in HepG2-sh3,HepG2-sh9,HepG2-sh11,HepG2-sh12,Hep3B-sh2 and Hep3B-sh3 clones.We selected HepG2-sh9,HepG2-sh11,Hep3B-sh2 and Hep3B-sh3 clones,and further verified by RT-PCR and western blot.The results showed that the mRNA expression levels and protein expression levels of CLDN6 in HepG2-sh9,HepG2-sh11,Hep3B-sh2 and Hep3B-sh3 were significantly lower than those in the no-load group,and the difference was statistically significant(P<0.05).The results of cellular immunofluorescence assay showed that the expression level of CLDN6 was decreased(P<0.05).Finally,HepG2-sh9(HepG2-sh-CLDN6)and Hep3B-sh3(Hep3B-sh-CLDN6)were selected for subsequent experiments.3.The effect of silence CLDN6 on proliferation,migration and invasion of human hepatoma carcinoma cells.The results of flow cytometry and CCK8 showed that the proliferation ability of HepG2-sh-CLDN6 and Hep3B-sh-CLDN6 cells decreased significantly compared with the no-load group,and the results were statistically significant(P<0.05,P<0.01).Wound-healing assay and transwell chamber experiment results showed that the migration ability of HepG2-sh-CLDN6 and Hep3B-sh-CLDN6 cells decreased significantly,and the results were statistically significant(P<0.05,P<0.01).Transwell chamber experiment results showed that the invasion ability of HepG2-sh-CLDN6 and Hep3B-sh-CLDN6 cells decreased significantly,and the results were statistically significant(P<0.01).4.Expression of EMT related proteins in human hepatoma carcinoma cells with CLDN6 gene silencing.Western blot results showed that the expression of E-cadherin increased in HepG2-sh-CLDN6 and Hep3B-sh-CLDN6 cells,and the expression of N-cadherin decreased,and the results were statistically significant(P< 0.05).Vimentin expression was decreased in HepG2-sh-CLDN6 and Hep3B-sh-CLDN6 cells,but not statistically significant.5.Expression of CLDN6 in human liver cancer tissues and its relationship with clinicopathological parameters.Immunohistochemical staining results showed that CLDN6 was mainly expressed in the cell membrane,and the positive expression rate was 79.17%.The results showed that the positive expression of CLDN6 in liver cancer tissues was related to the degree of tumor differentiation,which was not related to tumor stage,lymphatic metastasis,gender and age.Conclusion:1.CLDN6 is highly expressed in hepatoma carcinoma cells and mainly expressed in cell membrane.2.CLDN6 could enhance the proliferation,migration and invasion of hepatoma carcinoma cells and promote the occurrence of EMT in hepatoma carcinoma cells.3.CLDN6 is highly expressed in human liver cancer tissue,and its high expression is related to the degree of tumor differentiation,not concerned with tumor stage,lymphatic metastasis,gender and age.