miR-382通过PGC-1α对三阴性乳腺癌细胞株4T1生物学特性的影响

MiR-382 inhibits proliferation and migration of triple negative breast cancer cell line 4T1 by mediating PGC-1α

目的研究乳腺癌4T1细胞内miR-382下调PGC-1α对细胞生物学特性的影响。方法①以4T1细胞作为研究对象,miR-382mimics或抑制性探针anti-miR-382转染4T1细胞后,以qRT-PCR检测4T1中miR-382水平变化,qRT-PCR和Western blot检测各组PGC-1α的表达水平变化;②荧光素酶报告基因检测miR-382与PGC-1α3′UTR的结合位点;③将miR-382mimics或对照miR分别与pCDNA3.1或pCDNA-PGC1α共转染于4T1细胞,Transwell迁移实验检测细胞迁移能力的变化;CCK8实验检测细胞增殖活力的变化;皮下移植瘤实验和尾静脉肺移植瘤实验分别检测小鼠体内肿瘤生长及肺转移的变化。结果转染miR-382 mimics之后乳腺癌细胞PGC-1α表达明显降低(P<0.05),转染抑制性探针anti-miR-382后PGC-1α表达明显升高(P<0.05);miR-382与PGC-1α存在靶向调控核心序列,其作用位点位于PGC-1α3′UTR 565-582(核心序列ACAACTT)。相比对照+pCDNA3.1组,miR-382+pCDNA3.1组的细胞迁移和增殖活力明显降低,4T1皮下移植瘤体积和尾静脉移植瘤肺转移灶的面积显著减小(P<0.05);对照+pCDNA-PGC-1α较对照+pCDNA3.1组,细胞迁移和增殖活力明显提高,4T1皮下移植瘤体积和尾静脉移植瘤肺转移灶的面积显著增加(P<0.05);miR-382的抑瘤效应因PGC-1α过表达而被阻断。结论miR-382可能通过PGC-1α抑制4T1细胞株的增殖和肺转移能力,其作用位点可能位于PGC-1α3′UTR 565-582。

Objective To explore the effect of miR-382 down-regulating PGC-1αon the biological behaviors of triple negative breast cancer 4 T1 cells.Methods①After miR-382 mimics or inhibitory probe anti-miR-382 was transfected into 4 T1 cells,the expression level of miR-382 was detected by qRT-PCR.The changes of PGC-1αexpression at mRNA and protein levels were detected by qRT-PCR and Western blotting,respectively.②Luciferase reporter gene assay was adopted to identify the binding site of miR-382 mimics to PGC-1α3′UTR.③miR-382 mimics or control miR were co-transfected into 4 T1 cells with pCDNA3.1 or pCDNA-PGC1α,respectively.Transwell migration assay,CCK8 assay and subcutaneous xenografts and tail vein lung xenografts were respectively used to detect the changes in cell invasiveness,cell proliferation,and tumor growth and lung metastasis in mice.Results The expression of PGC-1αwas significantly decreased in 4 T1 cells after transfection with miR-382 mimics(P<0.05),and it was obviously increased after transfection with anti-miR-382(P<0.05).There was a targeted regulatory core sequence(ACAACTT)of miR-382 and PGC-1α,and the site was at PGC-1α3′UTR 565-582.Compared with the control+pCDNA3.1 group,the cell migration and proliferation activity of the miR-382+pCDNA3.1 group was significantly decreased,and the size of the 4 T1 subcutaneous tumor and the area of metastatic tumor in the lung were notably decreased(P<0.05).Compared with the control+pCDNA3.1 group,the cell migration and proliferation activity were significantly increased in the miR-382+pCDNA-PGC-1αgroup,and the size of 4 T1 subcutaneous tumor and the area of metastatic tumor in the lung was significantly increased(P<0.05);The antitumor effect of miR-382 was blocked by overexpression of PGC-1α.Conclusion MiR-382 inhibits the proliferation and lung metastasis of 4 T1 cells by down-regulating PGC-1α,and the interaction site may be PGC-1α3′UTR 565-582.