华蟾酥毒基通过调控circNFIX/miR-577通路抑制乳腺癌MDA-MB-231细胞的增殖、迁移和侵袭

Cinobufagin inhibits proliferation,migration and invasion of breast cancer MDA-MB-231 cells by regulating circNFIX/miR-577 pathway

目的:研究华蟾酥毒基(CnBu)对乳腺癌细胞MDA-MB-231恶性生物学行为的影响,并探讨其机制是否与调控环状RNA核因子IX(circNFIX)和miR-577表达相关。方法:将MDA-MB-231细胞分为对照(Con)组、CnBu-低剂量(L)组、CnBu-中剂量(M)组、CnBu-高剂量(H)组、si-NC组、si-circNFIX组、pcDNA组、pcDNA-circNFIX组、CnBu+pcDNA组、CnBu+pcDNAcircNFIX组。MTT、Transwell实验分析细胞增殖、迁移和侵袭能力,Westernblot分析Ki-67、基质金属蛋白酶2(MMP-2)和MMP-9蛋白表达。RT-qPCR分析circNFIX、miR-577表达量。双荧光素酶报告实验和RT-qPCR验证circNFIX和miR-577的靶向调控关系。结果:CnBu以剂量依赖方式下调Ki-67、MMP-2、MMP-9蛋白和circNFIX表达(P<0.05),上调miR-577表达(P<0.05),减弱MDA-MB-231细胞的增殖、迁移和侵袭能力(P<0.05)。miR-577与circNFIX序列直接结合,干扰或过表达circNFIX可显著提高或降低miR-577表达水平(P<0.05)。干扰circNFIX表达显著下调Ki-67、MMP-2、MMP-9蛋白表达(P<0.05),减弱MDA-MB-231细胞的增殖、迁移和侵袭能力(P<0.05)。过表达circNFIX显著减弱CnBu对MDA-MB-231细胞增殖、迁移、侵袭及相关蛋白表达的影响(P<0.05)。结论:CnBu可抑制乳腺癌MDA-MB-231细胞的增殖、迁移和侵袭,可能通过下调circNFIX/miR-577通路实现。

Objective:To investigate the effect of Cinobufagin(CnBu)on malignant biological behavior of breast cancer cell MDA-MB-231,as well as to explore whether its mechanism is related to regulation of circular RNA nuclear factor IX(circNFIX)and miR-577 expression.Methods:MDA-MB-231 cells were divided into control(Con)group,CnBu-low dose(L)group,CnBu-medium dose(M)group,CnBu-high dose(H)group,si-NC group,si-circNFIX group,pcDNA group,pcDNA-circNFIX group,CnBu+pcDNA group,CnBu+pcDNA-circNFIX group.MTT and Transwell experiment were used to analyze cell proliferation,migration and invasion ability,and Western blot was applied to analyze expressions of Ki-67,matrix metalloproteinase 2(MMP-2)and MMP-9 proteins.Expression levels of circNFIX and miR-577 were analyzed by RT-qPCR.Dual luciferase reporter experiment and RT-qPCR were selected to verify targeted regulation relationship between circNFIX and miR-577.Results:CnBu could down-regulate Ki-67,MMP-2,MMP-9 proteins and circNFIX expressions(P<0.05),up-regulate miR-577 expression(P<0.05),as well as reduced proliferation,migration and invasion ability of MDA-MB-231 cells(P<0.05),in a dose-dependent manner.miR-577 directly bound to circNFIX sequence,and interfered with circNFIX or circNFIX overexpression could significantly increase or decrease expression level of miR-577(P<0.05).Interference with circNFIX expression significantly down-regulated Ki-67,MMP-2,MMP-9 protein expressions(P<0.05),and reduced proliferation,migration and invasion ability of MDA-MB-231 cells(P<0.05).circNFIX overexpression significantly reduced the effect of CnBu on MDA-MB-231 cells proliferation,migration,invasion and related proteins expressions(P<0.05).Conclu⁃sion:CnBu can inhibit proliferation,migration and invasion of breast cancer MDA-MB-231 cells,which may be achieved by down-regulat‐ing circNFIX/miR-577 pathway.