Capn4 Enhances Osteopontin Expression Through Activation Of The Wnt/β-catenin Pathway To Promote Epithelial Ovarian Carcinoma Metastasis

Part Ⅰ Calpain small subunit-1(Capn4)is overexpressed in epithelial ovarian cancer tissues and cell linesObjective: To verify the expression of Capn4 in epithelial ovarian cancer(EOC)tissues and cell lines,and to analyze the relationship between Capn4 and the clinicopathologic characteristics of EOC.Methods: We examined the expression of Capn4 in normal ovarian epithelial tissues,borderline ovarian tumor tissues and EOC tissue specimens by an IHC assay.We examined the expression of Capn4 in a human normal ovarian cell line(Moody)and four ovarian cancer cell lines(Hey,Skov3,HO8910,HO8910-PM)by western blot.Results:(1)We found that the expression of Capn4 was higher in EOC tissues than in borderline ovarian tumor tissues and normal ovarian epithelial tissues.(2)Increased Capn4 expression was significantly correlated with lymph node metastasis(p = 0.009)and FIGO stage(p = 0.001).The relationship between Capn4 expression and the OS time of patients with EOC was analyzed by Kaplan–Meier analysis,revealing a significant correlation between high Capn4 expression and poor OS,compared with survival of patients with relatively low Capn4 expression.(3)We found that all of the cancer cell lines expressed higher levels of Capn4 protein compared with Moody cells.Conclusion:These findings confirm the overexpression of Capn4 in EOC tissues and cell lines,and that the increased expression of Capn4 predicts poor survival in EOC.Part Ⅱ Capn4 promotes the proliferation and migration of EOC cells in vitroObjective: To verify the effect of Capn4 on the proliferation,migration and cell cycle of EOC cells.Methods: We silenced Capn4 expression in the high Capn4-expressing cell line Hey by transfecting the cells with Capn4-siRNA,and overexpressed Capn4 in the low Capn4-expressing cell line HO8910 by transfecting the cells with pcDNA3.1-Capn4.To evaluate whether Capn4 affects the migratory abilities of EOC cells,we performed wound-healing and Transwell migration assays.To observe the effect of Capn4 expression on cell proliferation,we performed CCK-8 assay.Flow cytometry-based cell cycle analysis was used to analyze the effect of Capn4 on cell cycle.Results:(1)Overexpression of Capn4 increased cell proliferation in EOC cells.(2)Flow cytometry-based cell cycle analysis showed that Capn4 overexpression increased the percentage of cells in the S phase.(3)Capn4 promotes the migratory behavior of EOC cells in vitro.Conclusion: Capn4 promotes the migratory and proliferation behavior of EOC cells in vitro.Part Ⅲ Capn4 and OPN expression is positively correlatedObjective: To evaluate the relationship between Capn4 and OPN in EOC.Methods: We first analyzed the protein expression of Capn4 and OPN in consecutive sections of EOC clinical specimens by IHC analysis.Next,we investigated the expression of Capn4 and OPN in ovarian cancer cell lines.HO8910-PM cells are highly metastatic ovarian cancer cells,obtained by limiting-dilution cloning of the HO8910 cell line.Transwell migration assays was used to verify the migration ability of the two cell lines.The expression levels of Capn4 and OPN was examined by using western blot and immunofluorescent staining.To further verified the relationship between Capn4 and OPN,we increased the expression of Capn4 and decreased the OPN expression in the low Capn4-expressing cell line HO8910 by transfecting the cells with pcDNA3.1-Capn4 and si-OPN.Then we examined the expression of Capn4 and OPN by using Western blot,and the migration ability of ovarian cancer cells was abserved by Transwell assays.Results:(1)Capn4 and OPN expression is positively correlated in EOC tissues.(2)OPN and Capn4 expression are associated in ovarian cancer cell lines,and are related to cell migratory ability.(3)Enforced expression of Capn4 enhanced the migratory ability of HO8910 cells,whereas the siRNA-mediated downregulation of OPN significantly attenuated such Capn4-induced cell migration.Conclusion: Capn4 and OPN expression is positively correlated,and Capn4 may promote the metastasis of epithelial ovarian cancer cells by regulating the expression of OPN.Part Ⅳ Capn4 promotes OPN expression through the Wnt/βcatenin pathwayObjective: To identify the mechanism by which Capn4 upregulates OPN expression in the ovarian cancer cell lines.Methods: We evaluated the protein levels of b-catenin and OPN in Hey and HO8910 cells pretreated with either Capn4 siRNA or the Capn4-overexpression plasmid.We down-regulated the expression of b-catenin by siRNA,then examined the OPN expression.Down-regulating the expression of b-catenin at the same time of over-express the Capn4 protein level,using Western blot to examine the expression of OPN.To further analyze whether Wnt/βcatenin signaling is involved in the mechanism by which Capn4 promotes OPN expression,we treated the cells with the Wnt/βcatenin pathway activator LiCl and the inhibitor XAV939.Results: Capn4 promotes OPN expression through the Wnt/βcatenin pathway.Conclusion: In EOC cells,Capn4 promotes OPN expression through the Wnt/βcatenin pathway.