摘要
目的 探讨松果菊苷(ECH)调控miR-485-5p/三重基序蛋白7(TRIM7)信号通路对胶质母细胞瘤(GBM)细胞增殖、凋亡和迁移的影响.方法 qRT-PCR检测GBM细胞中miR-485-5p表达水平;筛选最佳干预细胞系(U251细胞),以不同浓度ECH(0、1、5、10、20 μmol·L-1)干预,MTT法检测各GBM细胞增殖情况;将U251细胞分为对照组(正常培养)、ECH组(10 μmol·L-1)、inhibitor-NC组(转染inhibitor-NC)、miR-485-5p inhibitor组(转染miR-485-5p inhibitor),检测细胞中miR-485-5p的表达水平;Edu染色、Transwell小室和流式细胞仪分别检测细胞增殖、迁移、侵袭和凋亡;Western blot法检测增殖细胞核抗原(PCNA)、基质金属蛋白酶-2(MMP-2)、TRIM7、E-cadherin、vimentin表达;双荧光素酶报告基因实验验证miR-485-5p和TRIM7的靶向关系.小鼠体内肿瘤形成实验验证ECH对GBM肿瘤生长及miR-485-5p/TRIM7的影响.结果 miR-485-5p在GBM细胞中表达水平降低(P<0.05);ECH显著抑制U251细胞增殖、迁移和侵袭,促进凋亡;降低U251细胞中PCNA、MMP-2、TRIM7、vimentin蛋白水平,升高E-cadherin蛋白水平(均P<0.05);抑制miR-485-5p表达可逆转ECH对U251细胞增殖、迁移和侵袭的抑制作用以及对凋亡的促进作用(均P<0.05).双荧光素酶报告基因实验证实miR-485-5p靶向调控TRIM7的表达(P<0.05);体内实验显示ECH可降低移植瘤质量和体积及TRIM7、Ki-67蛋白表达水平,增高移植瘤中miR-485-5p水平(P<0.05).结论 ECH可抑制GBM细胞增殖、迁移和侵袭,促进凋亡,可能与调控miR-485-5p/TRIM7信号通路有关.
Abstract
Aim To investigate the impacts of echinacoside(ECH)on the proliferation,apoptosis,and migration of glioblastoma(GBM)cells by regulating the miR-485-5p/triple motif protein 7(TRIM7)signaling pathway.Methods qRT-PCR was applied to detect the expression of miR-485-5p in GBM cells.The optimal intervention cell line(U251 cells)was screened to intervene with different concentrations of ECH(0,1,5,10,20 μmol·L-1).MTT method was applied to detect cell proliferation.U251 cells were divided into a control group(normal culture),a ECH group(10 μmol·L-1),a inhibitor-NC group(transfected with inhibitor NC),and a miR-485-5p inhibitor group(transfected with miR-485-5p inhibitor),The expression level of miR-485-5p in cells was detected.Edu staining,Transwell chamber,and flow cytometry were applied to detect cell proliferation,migration,invasion,and apoptosis,respectively.Western blot was applied to detect the expression of proliferating cell nuclear antigen(PCNA),matrix metalloproteinase-2(MMP-2),TRIM7,E-cadherin and vimentin.The double luciferase reporter gene experiment was applied to verify the targeting relationship between miR-485-5p and TRIM7.The tumor formation experiment in mice was applied to verify the effects of ECH on GBM tumor growth and miR-485-5p/TRIM7.Results The expression level of miR-485-5p decreased in GBM cells(P<0.05).ECH obviously inhibited the proliferation,migration,and invasion of U251 cells,promoted apoptosis,reduced the expression of PCNA,MMP-2,TRIM7,and vimentin proteins in U251 cells,and increased the expression of E-cadherin protein(P<0.05).Inhibiting the expression of miR-485-5p was able to reverse the inhibitory effects of ECH on U251 cell proliferation,migration,and invasion,and its promoting effect on apoptosis(P<0.05).The double luciferase reporter gene experiment confirmed that miR-485-5p targeted to regulate the expression of TRIM7(P<0.05).In vivo experiments showed that ECH was able to reduce the mass and volume of transplanted tumors,the expression levels of TRIM7 and Ki-67 proteins,and increased the level of miR-485-5p in transplanted tumors(P<0.05).Conclusion ECH can inhibit the proliferation,migration,and invasion of GBM cells,promote apoptosis,which may be related to the regulation of miR-485-5p/TRIM7 signaling pathway.
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