| Background and objectiveEndometrial cancer is one of the most common gynecological malignancies and has exhibited an increasing incidence rate in recent years.Although most of the patients diagnosis in the early stage,but there are still some special types of endometrial cancer is more aggressive.Some of advanced tumors still recur after primary surgery in metastatic disease.The main reason of poor prognosis in these patients is chemotherapy resistance.Therefore,they require novel biomarkers or targets identified for treating.Cancer stem cells(CSCs)which have the ability to self-renew and initiate tumors,represent a small population of cancer cells.CSCs are also responsible for tumor relapse after chemotherapy.SPARC-related modular calcium binding 2(SMOC-2),a matricellular protein,may mediate intercellular signaling and cell type-specific differentiation during gonad and reproductive tract development.Recently,SMOC-2 has been identified as the intestinal stem cell signature gene that is required for L1-mediated colon cancer progression.In this study,we investigated the role of SMOC-2 in endometrial cancer stem cells and molecular mechanism of SMOC-2 effects cell proliferation and chemotherapeutic resistance in endometrial cancer.Research methods1.Immunohistochemical method was used to detect the expression of SMOC-2 in endometrial carcinoma tissues.We also analysed the relationship between SMOC-2 expression and clinicopathological characteristics.2.In vivo and in vitro,the effects of SMOC-2 on the proliferation and chemotherapeutic resistance of endometrial cancer cells were detected by using siRNA or SMOC-2 plasmids to silence or overexpress SMOC-2.3.Endometrial cancer stem cells and non stem cells were isolated by flow cytometry and in vitro spheres assay.The expression levels of mRNA and protein of SMOC-2 in two groups were compared.After silencing of SMOC-2,we detected the clonogenic potential of endometrial cancer by sphere assay.We also collected the sphere cells and detected the expression of cancer stem cells related genes SOX2,OCT4 and NANOG by qPCR.Immunohistochemistry was used to detect the expression of SMOC-2 in endometrial carcinoma and whether it was consistent with the expression of CD 133 and CD44 in endometrial cancer stem cells.4.The signaling pathway that SMOC-2 can activate is screened by the luciferase reporter gene.5.The immunofluorescence and Western blotting method were used to further verify that SMOC-2 activates the WNT/?-catenin signaling pathway.6.The molecular mechanism of SMOC-2 activating the WNT/?-catenin signaling pathway was detected by CO-immunoprecipitation and co-culture immunoprecipitation.Research results1.The high expression rate of SMOC-2 in endometrial carcinoma was 73.25%,and the high expression of SMOC-2 was related to the histopathological classification,pathological classification and the depth of tumor myometrium infiltration.2.In vitro,silencing of SMOC-2 can increased the sensitivity of cell to contact inhibition and inhibit the proliferation of endometrial cancer cells.In contrast,overexpression of SMOC-2 can reduced the cell's sensitivity to contact inhibition and promote the proliferation of endometrial cancer cells.Silencing of SMOC-2 could increase the drug sensitivity of endometrial cancer cells to paclitaxel and cisplatin,and the cell survival rate was significantly lower than that of the control groups.In vivo,silencing of SMOC-2 can significantly increase the inhibitory rate of cisplatin on the tumor.3.The expression of SMOC-2 was higher in CD133+/CD44+cells than that in CD133/CD44-cells.SMOC-2 expression is greatly increased in spheres compared to monolayer cultures.Using the sphere assay,silencing SMOC-2 reduced the clonogenic potential of endometrial cancer cells and downregulated the expression of SOX2,OCT4,and NANOG,the stemness-associated genes.The expression of SMOC-2 in endometrial carcinoma is related to the markers of tumor stem cells,CD133 and CD44.4.The results of the luciferase reporter gene showed that silencing of SMOC-2 inhibited the WNT/?-catenin pathway,and overexpression of SMOC-2 activated WNT/?-catenin.Silencing of SMOC-2 also downregulated the expression of WNT/?-catenin target gene.The results of immunofluorescence and Western blotting showed that the expression of ?-catenin in the nucleus increased after overexpression of SMOC-2.5.SMOC-2 can directly interact with the receptors Fzd6 and LRP6 of the Wnt pathway and promote the binding between the ligands and the receptors(Wnt3a and Wnt10b).Research conclusions1.SMOC-2 can promote the proliferation and drug resistance of endometrial cancer cells.2.SMOC-2 is a novel signature gene of endometrial cancer stem cells.3.SMOC-2 activates the WNT/?-catenin pathway in endometrial carcinoma and promotes the nucleation of ?-catenin.4.SMOC-2 physically interacted with FZD6 and LRP6,enhanced their interaction with canonical WNT ligands and thus activated the WNT/?-catenin pathway.5.SMOC-2 may be a new method for the treatment of endometrial cancer by targeting cancer stem cells. |
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