CCL20/CCR6/Notch1信号通路影响卵巢癌CD44^(+)/CD117^(+)细胞亚群增殖能力和耐药性的机制

The molecular mechanism of the CCL20/CCR6/Notch1signaling pathway affecting the proliferation ability and drug resistance of ovarian cancer CD44^(+)/CD117^(+)cell subpopulations

目的探究CCL20/CCR6/Notch1信号通路影响卵巢癌CD44^(+)/CD117^(+)细胞亚群增殖能力和耐药性的机制。方法离体培养人EOC SKOV-3细胞系,CD44^(+)CD117^(+)CSC随机分为对照组和紫杉醇组。对照组:在具有CM或培养基的96孔板中培养卵巢癌细胞(4000/孔),添加生理盐水培养24 h。紫杉醇组:在具有CM或培养基的96孔板中培养卵巢癌细胞(4000/孔),添加紫杉醇100μg培养24 h。蛋白质印迹法检测CD44^(+)CD117^(+)CSCsCCL20、CCR6、Notch1以及间充质标记物N-钙黏蛋白和Snail的表达;细胞迁移实验和细胞侵袭实验检测CD44^(+)CD117^(+)CSCs的迁移和侵袭能力;流式细胞仪分析CD44^(+)CD117^(+)CSCs的凋亡水平;细胞集落形成实验分析CD44^(+)CD117^(+)CSCs的增殖能力。结果与对照组相比,紫杉醇组CD44^(+)CD117^(+)CSCs细胞中CCL20、CCR6和Notch1表达上调,间充质标记物N-钙黏蛋白和Snail蛋白上调,上皮标记物E-钙黏蛋白下调,差异有统计学意义(P<0.05)。与对照组相比,紫杉醇组CD44^(+)CD117^(+)CSCs细胞中的迁移侵袭能力上调,紫杉醇组CD44^(+)CD117^(+)CSCs细胞中的凋亡水平下调,紫杉醇处理CD44^(+)CD117^(+)CSCs细胞中的细胞集落形成数目水平上调,差异有统计学意义(P<0.05)。结论卵巢癌CSCs自分泌CCL20并通过与CCR6受体结合活化Notch1信号通路,进而上调IL-1Β的表达并维持侵袭转移特性和耐药性。

Objective Explore the mechanism by which the CCL20/CCR6/Notch1signaling pathway affects the proliferation ability and drug resistance of ovarian cancer CD44^(+)/CD117^(+)cell subsets.Methods Human EOC SKOV-3 cells were cultured in vitro,and CD44^(+)CD117^(+)CSC cells were randomly divided into control group and paclitaxel group.Control group:Ovarian cancer cells were cultured in 96-well plates with CM or medium(4000/well)and cultured with normal saline for 24h.Paclitaxel group:Ovarian cancer cells were cultured in 96-well plates with CM or medium(4000/well),and paclitaxel was added 100μg for 24h.The expressions of CD44^(+)CD117^(+)CSCsCCL20,CCR6,Notch1 and mesenchymal markers N-cadherin and Snail were detected by western blotting.The migration and invasion ability of CD44^(+)CD117^(+)CSCs were detected by cell migration and invasion assay.The apoptosis level of CD44^(+)CD117^(+)CSCs was analyzed by flow cytometry.The proliferation capacity of CD44^(+)CD117^(+)CSCs was analyzed by cell colony formation assay.Results Compared with the control group,the upregulation of CCL20,CCR6,and Notch1in CD44^(+)CD117^(+)CSCs cells in the paclitaxel group,the upregulation of mesenchymal markers N-cadherin and Snail protein in CD44^(+)CD117^(+)CSCs cells in the paclitaxel group,while the downregulation of epithelial marker E-cadherin were statistically significant(P<0.05).Compared with the control group,the migration and invasion ability of CD44^(+)CD117^(+)CSCs cells in the paclitaxel group was upregulated,the apoptosis level in CD44^(+)CD117^(+)CSCs cells in the paclitaxel group was down regulated,the level of cell colony formation in CD44^(+)CD117^(+)CSCs cells treated with paclitaxel was upregulated,and the difference were statistically significant(P<0.05).Conclusion Ovarian cancer CSCs secrete CCL20and activate the Notch1signaling pathway by binding to CCR6receptors,thereby upregulating IL-1Βexpres-sion and maintenance of invasion and metastasis characteristics and drug resistance.