苦参碱通过miR-17-5p/MKNK2分子轴抑制膀胱癌干细胞增殖和侵袭

Matrine inhibits the proliferation and invasion of bladder cancer stem cells by regulating the miR-17-5p/MKNK2 molecular axis

目的通过miR-17-5p/MKNK2分子轴研究苦参碱(atrine,MT)对膀胱癌干细胞增殖和侵袭的影响,并阐明其作用机制。方法采用流式细胞术分选膀胱癌干细胞(transitional cell carcinoma cells,TCCSUP),并用干细胞成球实验检测不同剂量MT处理的膀胱癌干细胞的成球能力,采用CCK-8和Transwell检测膀胱癌干细胞增殖和侵袭,采用RT-qPCR检测膀胱癌干细胞中miRNAs的表达水平,采用Western blot检测MKNK2的表达水平,采用双荧光素酶报告基因实验验证miR-17-5p和MKNK2的靶向关系。结果通过分选出的TCCSUP细胞在显微镜下观察发现,细胞生长状态良好,呈球形或椭圆形,细胞膜边界清晰;CCK-8、Transwell和干细胞成球实验检测结果显示,MT可呈浓度依赖性显著抑制膀胱癌干细胞增殖、侵袭以及成球能力,TCCSUP干细胞半数抑制浓度为2 mg·L^(-1)。miR-17-5p在膀胱癌干细胞内异常高表达,MT可显著下调miR-17-5p的表达水平;过表达miR-17-5p可明显促进膀胱癌干细胞的增殖、侵袭以及干细胞成球;同时过表达miR-17-5p和MKNK2可显著MKNK2下调过表达MKNK2对TCCSUP干细胞增殖、侵袭和干细胞成球的抑制作用。结论MT通过下调miR-17-5p,促进MKNK2的表达,抑制TCCSUP干细胞增殖、侵袭和成球能力。

Objective To study the effect of MT on the proliferation and invasion of bladder cancer stem cells through the molecular axis of miR-17-5 p/MKNK2,and to elucidate its mechanism of action.Methods Bladder cancer stem cells were sorted by flow cytometry,and the globule ability of bladder cancer stem cells treated with different doses of MT were detected by stem cell spheroid assay.CCK-8 and Transwell were used to detect the proliferation and invasion of bladder cancer stem cells.The expression levels of miRNA in bladder cancer stem cells were detected by RT-qPCR.The expression level of MKNK2 was detected by Western blot.The dual luciferase reporter gene assay confirmed the targeting relationship between miR-17-5 p and MKNK2.Results The selected TCCSUP stem cells were observed under the microscope and found to be in good growth condition,spherical or elliptic,with clear membrane boundary.CCK-8,Transwell and stem cell pellet-forming assay showed that MT significantly inhibited the proliferation,invasion and pellet-forming ability of bladder cancer stem cells in a concentration-dependent manner,and 2 mg·L^(-1)MT was half of the inhibition concentration of TCCSUP stem cells.The expression of miR-17-5 p was abnormally high in bladder cancer stem cells,and MT could significantly down-regulate the expression level of miR-17-5 p.Overexpression of miR-17-5 p can significantly promote proliferation,invasion and pellet-forming of bladder cancer stem cells.Overexpression of miR-17-5 p and MKNK2 at the same time can significantly down-regulate the inhibitory effects of MKNK2 overexpression on TCCSUP stem cell proliferation,invasion and stem cell spheronization.Conclusion MT can inhibit the proliferation and invasion ability of TCCSUP stem cells by up-regulating the expression of MKNK2 and down-regulating the expression level of miR-17-5 p.