槐定碱通过调节MCP-1/CCR2信号轴抑制膀胱癌恶性进展的实验研究

Experimental study on sophoridine inhibiting malignant progression of bladder cancer by regulating MCP-1/CCR2 signal axis

目的:探究槐定碱对膀胱癌恶性进展的影响以及该过程中对MCP-1/CCR2信号轴的调控机制。方法:通过CCK-8检测槐定碱对膀胱癌细胞5637的半数抑制浓度,随后将细胞分为对照组、槐定碱低浓度组、槐定碱高浓度组、槐定碱高+pcDNA组、槐定碱高+pcDNA-MCP-1组。CCK-8检测各组细胞的增殖活性;流式细胞术检测各组细胞凋亡情况和细胞周期;Transwell实验检测各组细胞迁移和侵袭能力;Western blot检测各组细胞中E-cadherin、Vimentin、N-cadherin、MCP-1、CCR2蛋白的表达;qRT-PCR检测各组细胞中MCP-1、CCR2 mRNA的水平;建立移植瘤裸鼠模型,观察肿瘤生长情况并检测肿瘤组织中MCP-1、CCR2 mRNA及蛋白水平。结果:与对照组相比,槐定碱低、高浓度组细胞的存活率、克隆形成数量、S期细胞比例、迁移和侵袭细胞数量、Vimentin和N-cadherin表达、MCP-1和CCR2 mRNA及蛋白的水平均降低(P<0.05),细胞凋亡率、G2/M期细胞比例、E-cadherin表达升高(P<0.05);与槐定碱高浓度组相比,槐定碱高+pcDNA-MCP-1组细胞的存活率、克隆形成数量、S期细胞比例、迁移和侵袭细胞数量、Vimentin和N-cadherin表达、MCP-1和CCR2 mRNA及蛋白的水平均升高(P<0.05),细胞凋亡率、G2/M期细胞比例、E-cadherin表达降低(P<0.05);裸鼠体内移植瘤实验结果显示,与对照组相比,槐定碱组小鼠肿瘤组织的重量和体积减小,MCP-1和CCR2 mRNA及蛋白水平降低(P<0.05);与槐定碱组相比,槐定碱+pcDNA-MCP-1组小鼠肿瘤组织的重量和体积增加,抑瘤率减小,MCP-1和CCR2 mRNA及蛋白水平升高(P<0.05)。结论:槐定碱可能通过抑制MCP-1/CCR2信号轴来抑制膀胱癌的恶性进展。

Objective:To explore the impact of sophoridine on the malignant progression of bladder cancer and its regulation mechanism on MCP-1/CCR2 signal axis in this process.Methods:CCK-8 was applied to detect the half inhibitory concentration of sophoridine on bladder cancer cells 5637,and then the cells were separated into control group,low concentration sophoridine group,high concentration sophoridine group,high concentration sophoridine+pcDNA group,and high concentration sophoridine+pcDNA-MCP-1 group.CCK-8 sophoridine was applied to detect the proliferation activity of cells in each group.Flow cytometry was applied to detect cell apoptosis and cell cycle in each group.Transwell test was applied to detect cell migration and invasion abilities in each group.Western blot was applied to detect the expression of E-cadherin,Vimentin,and N-cadherin proteins of cells in each group.qRT-PCR was applied to detect the levels of MCP-1 and CCR2 mRNA of cells in each group.The nude mouse model of transplanted tumors was established,the tumor growth was observed,and the mRNA and protein levels of MCP-1 and CCR2 in tumor tissues were detected.Results:Compared with the control group,the cell survival rate,number of clones formed,proportion of S phase cells,numbers of migrating and invading cells,expression of Vimentin and N-cadherin,and levels of MCP-1 and CCR2 mRNA and protein in the low and high concentration sophoridine groups reduced(P<0.05),the apoptosis rate,proportion of G2/M phase cells,and expression of E-cadherin increased(P<0.05).Compared with the high concentration sophoridine group,the cell survival rate,number of clones formed,proportion of S phase cells,numbers of migrating and invading cells,expression of Vimentin and N-cadherin,and levels of MCP-1 and CCR2 mRNA and protein in the high concentration sophoridine+pcDNA-MCP-1 group increased(P<0.05),the apoptosis rate,proportion of G2/M phase cells,and expression of E-cadherin reduced(P<0.05).The results of in vivo tumor transplantation experiment showed that compared with the control group,the weight and volume of tumor tissue in the sophoridine group decreased,and the mRNA and protein levels of MCP-1 and CCR2 decreased(P<0.05).Compared with the sophoridine group,the weight and volume of tumor tissue in the sophoridine+pcDNA-MCP-1 group increased,while the tumor inhibition rate decreased,and the mRNA and protein levels of MCP-1 and CCR2 increased(P<0.05).Conclusion:Sophoridine may inhibit the malignant progression of bladder cancer by inhibiting the MCP-1/CCR2 signal axis.