Study On The Effect Of LGR6 On The Sensitivity Of Ovarian Epithelial Cancer Cells To Chemotherapy

BackgroundOvarian cancer is one of the leading causes of cancer-related deaths in women,due to the recurrent relapse and platinum resistance.Existence of cancer stem cells has been identified to significantly contribute to the early recurrence and chemoresistance in multiple human cancers,including ovarian cancer.Clarifying the potential mechanism of inducing and maintaining "cancer stem cell-like"characteristics would improve the chemotherapy efficacy and prognosis in ovarian cancer patients.Continuous activation of Wnt/catenin pathway is critical to maintain the characteristics of cancer stem cells in various types of tumors.LGR6,a leucine-rich G protein-coupled receptor,plays an important role in activating classical Wnt by binding its ligands.However,the clinical significance,biological function in ovarian cancer,as well as the underlying mechanism of LGR6 in regulating the activity of Wnt/β-catenin signaling remain unclear.ObjectiveTo investigate the clinical significance of LGR6 in ovarian cancer,the effect of LGR6 on stem cell-like characteristics and chemosensitivity on ovarian cancer cells,and the effect of LGR6 on the activity of Wnt/β-catenin signaling in ovarian cancer cells.Methods1.The expression levels and clinical significance of LGR6 in ovarian cancer was investigated with quantitative real-time PCR,Western blotting and immunohistochemistry.2.Stable LGR6-downexpressing ovarian cancer cell lines were constructed.The role of LGR6 in inducing and maintaining cancer stem cell characteristics in ovarian cancer was examined by spheroid formation assay,side population analysis and the expression levels of stem cell factors via flow cytometry and fluorescence quantitative PCR.3.The effect of LGR6 on cell apoptotic ability of ovarian cancer cells was detected by CCK-8 method and AnnexinV-FITC/PI double staining in order to determine the sensitivity of ovarian cancer cells to cisplatin and paclitaxel.Mitochondrial membrane potential was measured by JC-1 staining.The expression of apoptosis-related proteins and genes were detected by Caspase-9/-3 substrate activity assay and Western blotting.An animal model of tumorigenesis in vivo was constructed to evaluate the effect of silencing LGR6 on the efficacy of cisplatin to ovarian cancer cells in vivo.4.Western blotting was used to analyze the nuclear entry of(3-catenin in the cell model.The transcriptional activity of TCF was detected by double luciferase reporter gene detection system.The downstream gene expression of Wnt pathway was detected by fluorescence quantitative PCR.The effect of silencing LGR6 on the activation of Wnt/β-catenin pathway was explored.C33Y mutant p-catenin was replenished in the cell model to identify that Wnt/β-catenin participated in the maintenance of stem cell characteristics and chemosensitivity of ovarian cancer cells induced by LGR6.ResultsLGR6 was up-regulated in ovarian cancer tissues,with the highest levels in high-grade serous ovarian cancer.High expression of LGR6 was positively correlated with FIGO staging and poor efficacy of chemotherapy in ovarian cancer,and negatively correlated with overall survival rate and progression-free survival rate.Silencing LGR6 inhibited the characteristics of ovarian cancer stem cells and improved the sensitivity of ovarian cancer cells to chemotherapeutic drugs in vitro and in vivo.Further investigation revealed that silencing LGR6 repressed stem cell characteristics and enhanced chemosensitivity of ovarian cancer cells via inhibiting classical Wnt pathway in ovarian cancer cells.ConclusionOur results indicate that LGR6 induces and maintains the "stem-like"characteristics of ovarian cancer cells by activating Wnt/β-catenin pathway,which further promotes chemo-resistance of ovarian cancer cells to cisplatin.These findings support the idea that LGR6 may serve as a potential prognostic marker in patients with ovarian cancer,as well as might be used as a potential therapeutic target for drug-resistant ovarian cancer.