miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究

Effects of miR-34a-5p on Triple Negative Breast Cancer Cells and Related Mechanisms

目的:探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法:利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果:TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论:miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。

Objective:To investigate the expression of miR-34a-5p in triple negative breast cancer(TNBC),its role in TNBC cell proliferation,apoptosis and migration,its effects on tumor growth in TNBC mice and its impact on the expression of B7-H1 in TNBC.Methods:RT-qPCR and Western blot were used to analyze the expression of miR-34a-5p and B7-H1 in TNBC cells,respectively.Kaplan-Meier was used to analyze the association between the expressions of miR-34a and B7-H1 with the survival rates of TNBC patients.The effects of miR-34a-5p on the proliferation,apoptosis and migration of TNBC cells were detected by CCK-8,flow cytometry and wound healing.The interaction between miR-34a-5p and B7-H1 was verified by Dual-luciferase reporter assays.The expression levels of miR-34a-5p and B7-H1 were detected by RT-qPCR and Western blot after transfected with miR-34a-5p mimics into TNBC cells.RT-qPCR,Western blot and IHC were used to detect the effect of miR-34a-5p agomir on the expression of miR-34a-5p and B7-H1 in MDA-MB-231 tumor bearing mice.Results:The expression level of miR-34a-5p in TNBC cells was lower than that in normal breast cells,and the expression level of B7-H1 was higher than that in normal breast cells,low expression of miR-34a and high expression of B7-H1 were related to the poor prognosis of TNBC patients,and the difference was statistically significant(P<0.01).miR-34a-5p could inhibit cell proliferation and migration,promote cell apoptosis,and target downregulate the expression of B7-H1 in TNBC cells.miR-34a-5p agomir inhibited tumor growth and B7-H1 expression of MDA-MB-231 tumor bearing mice.Conclusion:miR-34a-5p plays an important role in the development and progression of TNBC.Targeting miR-34a-5p/B7-H1 may become a new treatment strategy for TNBC patients.