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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (1): 18-26    DOI: 10.13523/j.cb.2206036
研究报告     
miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*
邓思雨1,梁冰1,2,魏薇1,王孟娜1,曹友德1,2,**()
1 重庆医科大学基础医学院 重庆 400016
2 重庆医科大学附属第一医院 重庆 400016
Effects of miR-34a-5p on Triple Negative Breast Cancer Cells and Related Mechanisms
DENG Si-yu1,LIANG Bing1,2,WEI Wei1,WANG Meng-na1,CAO You-de1,2,**()
1 College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China
2 The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
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摘要：

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。
关键词： microRNA-34a-5p;  三阴性乳腺癌;  B7同源体1    
Abstract:

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.
Key words: MicroRNA-34a-5p    Triple negative breast cancer    B7 homolog 1
收稿日期: 2022-06-19 出版日期: 2023-02-14
ZTFLH:  R737  
基金资助: *国家自然科学基金(82102909)
通讯作者: **曹友德 电子信箱:420306100@qq.com   
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引用本文:

邓思雨, 梁冰, 魏薇, 王孟娜, 曹友德. miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*[J]. 中国生物工程杂志, 2023, 43(1): 18-26.

DENG Si-yu, LIANG Bing, WEI Wei, WANG Meng-na, CAO You-de. Effects of miR-34a-5p on Triple Negative Breast Cancer Cells and Related Mechanisms. China Biotechnology, 2023, 43(1): 18-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2206036    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I1/18

引物 序列(5'→3')
U6上游 5'-TGGAACGCTTCACGAATTTGCG-3'
U6下游 5'-GGAACGATACAGAGAAGATTAGC-3'
miR-34a茎环 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAACC-3'
miR-34a-5p上游 5'-CGCGTGGCAGTGTCTTAGCT-3'
miR-34a-5p下游 5'-AGTGCAGGGTCCGAGGTATT-3'
表1  引物序列
图1  miR-34a-5p在MCF 10A、MDA-MB-231、BT-549细胞株中表达的差异及与预后的相关性
图2  miR-34a-5p对TNBC细胞的增殖、凋亡、迁移的影响
图3  miR-34a-5p与B7-H1的靶向关系
图4  转染miR-34a-5p抑制TNBC细胞中B7-H1的表达
图5  miR-34a-5p抑制TNBC荷瘤小鼠的肿瘤生长并下调B7-H1表达
图6  B7-H1在乳腺癌中与免疫细胞的关联性
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