Research On The Overexpression Of CLDN6 In Inhibiting Lung Metastasis Of Breast Cancer

Breast cancer is the most common malignant tumor in women all over the world,which is prone to lung metastasis.The occurrence of lung metastasis of breast cancer is affected by many factors,among which the tight junction protein family plays an important role,but its specific regulatory mechanism is not clear.Claudins(CLDNs)is the main transmembrane protein in tight junction,which can exert the function of cell barrier,and its abnormality can change the structure or function of tight junction.CLDN6,a member of the CLDNs family,has been confirmed to inhibit the proliferation,migration and invasion of tumor cells,but its role in lung metastasis of breast cancer remains to be further confirmed.Therefore,in this study,breast cancer 4T1 cells stably overexpressing CLDN6 were obtained by lentivirus transfection and puromycin screening,and the effects of overexpression of CLDN6 on proliferation,migration and apoptosis of breast cancer 4T1 cells and its possible mechanism were detected at the cell level.In order to provide experimental basis for the prevention and treatment of lung metastasis of breast cancer,the general state,the changes of body weight and the number of pulmonary nodules and the possible regulatory mechanism of overexpressed CLDN6 in mice with lung metastasis of breast cancer were detected at animal level.In the cell experiment,the low expression of CLDN6 in 4T1 cells was confirmed by qPCR and Western Blot results,and the breast cancer 4T1 cells stably overexpressing CLDN6 were successfully transfected and screened.The cell experiment was divided into three groups: breast cancer 4T1 cells in Control group,breast cancer 4T1 cells transfected with empty vector in Vector group,and breast cancer 4T1 cells stably overexpressing CLDN6 in CLDN6 group.CCK-8 assay,plate colony formation assay and flow cytometry were used to detect the effect of CLDN6 overexpression on the proliferation of 4T1 cells.The results showed that the proliferation rate of 4T1 cells was slowed down,the clone formation ability was significantly decreased,and the cell cycle was changed after transfection of CLDN6.Cell scratch assay was used to detect the effect of overexpression of CLDN6 on the migration ability of4T1 cells.The results showed that overexpression of CLDN6 inhibited the migration ability of 4T1 cells.By measuring the transepithelial resistance of monolayer cells and the permeability coefficient of FITC-bovine serum albumin,it was found that overexpression of CLDN6 enhanced the structure and function of tight junction between 4T1 cells.The results of Hoechst33342 and Di D staining showed that overexpression of CLDN6 induced apoptosis of 4T1 cells.Finally,the results of Western Blot detection showed that the effects of overexpression of CLDN6 on the proliferation,migration and apoptosis of breast cancer 4T1 cells may be related to the activation of p38 MAPK signal pathway.In the animal experiment,18 BALB/c female mice were randomly divided into Control group,Vector group and CLDN6 group.Breast cancer 4T1 cell suspension was injected intravenously in Control group,4T1 cell suspension transfected with empty vector was injected in Vector group,and 4T1 cell suspension stably overexpressing CLDN6 was injected in CLDN6 group.Since the establishment of the model,the body weight of mice was recorded every day.On the 30 th day,the mice were killed to collect lung tissue,and the total number of metastatic nodules on the lung surface was recorded and pathological examination was made.At the same time,Western Blot was used to detect the protein expression levels of ASK1,p-ASK1,p38 MAPK and p-p38 MAPK in the lung tissue of mice in each group.After three weeks of modeling,the diet and drinking water movement of mice in Control group and Vector group were slow,the activity decreased,the response to the outside world was slow,the hair was rough and without luster,while the CLDN6 group was more sensitive to the outside world,and the hair was smooth and shiny.At the fifth week of modeling,the weight loss of mice in CLDN6 group was significantly lower than that in Control group and Vector group,indicating that overexpression of CLDN6 could inhibit the weight loss of model mice.When the mice were killed to collect lung tissue,the number of metastatic nodules on the lung surface of CLDN6 group decreased significantly.Combined with the calculation of the total number of metastatic nodules on the lung surface,it was confirmed that overexpression of CLDN6 could inhibit lung metastasis of breast cancer in mice.The results of Western Blot detection showed that the expression of p-p38MAPK/p38 MAPK in CLDN6 group was significantly higher than that in Control group and Vector group,and p-ASK1/ASK1 was significantly lower than that in Control group(phosphorylation inactivation).These results further prove that the inhibition of lung metastasis of mouse breast cancer 4T1 cells by overexpression of CLDN6 may be related to the activation of ASK1/p38 MAPK signal pathway.To sum up,overexpression of CLDN6 can inhibit the proliferation and migration of breast cancer 4T1 cells,enhance the function of tight junction between cells,and induce apoptosis.The inhibitory effect of overexpression of CLDN6 on lung metastasis of mouse breast cancer 4T1 cells may be related to the regulation of ASK1/p38 MAPK signal pathway.