水飞蓟素通过调控miR-124-3p/WEE1轴影响胶质瘤细胞恶性生长的机制研究

Mechanism of silymarin on malignant growth of glioma cells by regulating miR-124-3p/WEE1 axis

目的探讨水飞蓟素(SM)对胶质瘤细胞恶性生长的影响以及对miR-124-3p/WEE1轴的调控机制。方法将胶质瘤U87细胞分为对照组,SM低、中、高浓度组,SM高浓度+miR-124-3p抑制剂组(SM高+miR-124-3p inhibitor组)。采用CCK-8法检测细胞增殖活性,Transwell实验检测细胞迁移和侵袭能力,流式细胞术检测细胞周期变化,Western blotting检测细胞中细胞周期蛋白D1(cyclin D1)及凋亡相关蛋白的表达,实时定量PCR检测细胞中miR-124-3p和WEE1 mRNA水平,荧光素酶活性实验验证miR-124-3p和WEE1之间的靶向关系,建立NOD/SCID小鼠颅内移植瘤模型并进行给药和分析。结果与对照组比较,不同浓度SM处理组的细胞增殖活性、迁移和侵袭细胞数、cyclin D1蛋白表达、WEE1 mRNA表达水平降低,G0/G1周期细胞数、cleaved caspase-8、cleaved caspase-9、cleaved caspase-3及miR-124-3p表达升高(P<0.05);进一步转染miR-124-3p inhibitor后发现,SM对胶质瘤细胞恶性行为的抑制作用被逆转。小鼠体内实验显示,SM处理组肿瘤的质量和体积均低于模型组(P<0.05),且小鼠体质量无显著变化(P>0.05)。结论SM可通过上调miR-124-3p靶向下调WEE1抑制胶质瘤细胞的恶性生长。

Objective To investigate the impact of silymarin(SM)on the malignant growth of glioma cells and the regulatory mechanism on the miR-124-3p/WEE1 axis.Methods Glioma U87 cells were grouped into control,SM low,medium,and high concentration groups,and SM high concentration+miR-124-3p inhibitor group(SM high+miR-124-3p inhibitor group).CCK-8 was used to measure the proliferation rate of cells;Transwell^(■)assay was applied to assay the migration and invasion of cells;cell cycle progression was detected by flow cytometry;Western blotting was applied to measure the expression of cyclin D1 and apoptosis-related proteins;the levels of miR-124-3p and WEE1 mRNA were determined by qRT-PCR;and a luciferase activity test was applied to verify the targeting relationship between miR-124-3p and WEE1;in addition,the establishment,administration,and analysis of a NOD/SCID mouse model of intracranial transplanted tumor were conducted.Results Compared with the control group,the cell proliferation,the numbers of migrating and invading cells,the expression of cyclin D1,and the level of WEE1 mRNA in the various SM treatment groups decreased,the number of cells in G0/G1 phase,the expression of cleaved caspase-8,cleaved caspase-9,cleaved caspase-3 and miR-124-3p increased(P<0.05);furthermore,transfection of miR-124-3p inhibitor reversed the inhibitory effect of SM on the malignant behavior of glioma cells.In vivo experiments with mice showed that the weights and volumes of tumors in the SM treatment group were lower than those in the model group(P<0.05),and there was no discernible change in the weight of the mice(P>0.05).Conclusion SM can inhibit the malignant growth of glioma cells by upregulating miR-124-3p and downregulating WEE1.