长链非编码RNA MAFG-AS1敲低通过MAFG-AS1/miR-3196/TFAP2A轴使JAK2/STAT3信号通路失活，从而阻断乳腺癌细胞的恶性进展。
Long non-coding RNA MAFG-AS1 knockdown blocks malignant progression in breast cancer cells by inactivating JAK2/STAT3 signaling pathway via MAFG-AS1/miR-3196/TFAP2A axis.
作者信息
Ding Mingxing, Fu Yongqiang, Guo Fangming, Chen Haohao, Fu Xiaoyan, Tan Wenzhuang, Zhang Hui
机构信息
Medical Molecular Biology Laboratory, Medical College, Jinhua Polytechnic Jinhua, Zhejiang, China.
Department of Laboratory Animals Center, Jinhua Institute for Food and Drug Control Jinhua, Zhejiang, China.
出版信息
Int J Clin Exp Pathol. 2020 Oct 1;13(10):2455-2473. eCollection 2020.
BACKGROUND
Breast cancer is still a leading threat to women's lives. Long non-coding RNAs (lncRNA) associated with cancer progression are getting attention. The objective of this study was to investigate the role of lncRNA MAFG-antisense 1 (MAFG-AS1) and mechanisms of action in breast cancer.
METHODS
The expression of MAFG-AS1, microRNA-3196 (miR-3196) and transcription factor AP-2 alpha (TFAP2A) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cell proliferation was assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The number of colonies was observed through colony formation assay. The protein levels of Cyclin D1, Ki67, Bcl-2 associated X protein (Bax), B-cell lymphoma2 (Bcl-2), Hexokinase II (HK2), lactate dehydrogenase A (LDHA), TFAP2A, Janus kinase 2 (JAK2), phosphorylated-JAK2 (p-JAK2), signal transducer and activator of transcription 3 (STAT3), and phosphorylated-STAT3 were quantified by western blot. The cell apoptosis was monitored using flow cytometry. The glycolysis progression was evaluated according to glucose consumption and lactate production. The relationship between miR-3196 and MAFG-AS1 or TFAP2A was predicted by the online tool starBase and verified by the dual-luciferase reporter assay. The role of MAFG-AS1 was determined by the tumor formation assay in nude mice.
RESULTS
MAFG-AS1 was highly expressed in tumor tissues and cells. MAFG-AS1 knockdown restrained proliferation, colony formation, and glycolysis but promoted apoptosis of breast cancer cells. MiR-3196 was a target of MAFG-AS1, and its inhibition reversed the role of MAFG-AS1 knockdown. TFAP2A was a target of miR-3196, and its overexpression abolished the effects of miR-3196 reintroduction. MAFG-AS1 knockdown suppressed the activity of the JAK2/STAT3 signaling pathway. Moreover, MAFG-AS1 knockdown reduced tumor growth .
CONCLUSION
MAFG-AS1 knockdown attenuated breast cancer progression and through activation of the JAK2/STAT3 signaling pathway by the MAFG-AS1/miR-3196/TFAP2A regulatory axis.
背景
乳腺癌仍然是对女性生命的主要威胁。与癌症进展相关的长链非编码RNA（lncRNA）正受到关注。本研究的目的是探讨lncRNA MAFG反义1（MAFG-AS1）在乳腺癌中的作用及其作用机制。
方法
采用定量实时聚合酶链反应（qRT-PCR）检测MAFG-AS1、微小RNA-3196（miR-3196）和转录因子AP-2α（TFAP2A）的表达。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四氮唑溴盐（MTT）法评估细胞增殖。通过集落形成试验观察集落数量。采用蛋白质印迹法检测细胞周期蛋白D1、Ki67、Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤2（Bcl-2）、己糖激酶II（HK2）、乳酸脱氢酶A（LDHA）、TFAP2A、Janus激酶2（JAK2）、磷酸化JAK2（p-JAK2）、信号转导子和转录激活子3（STAT3）以及磷酸化STAT3的蛋白水平。采用流式细胞术监测细胞凋亡。根据葡萄糖消耗和乳酸生成评估糖酵解进程。通过在线工具starBase预测miR-3196与MAFG-AS1或TFAP2A之间的关系，并通过双荧光素酶报告基因试验进行验证。通过裸鼠成瘤试验确定MAFG-AS1的作用。
结果
MAFG-AS1在肿瘤组织和细胞中高表达。敲低MAFG-AS1可抑制乳腺癌细胞的增殖、集落形成和糖酵解，但促进其凋亡。miR-3196是MAFG-AS1的靶标，抑制miR-3196可逆转敲低MAFG-AS1的作用。TFAP2A是miR-3196的靶标，过表达TFAP2A可消除重新导入miR-3196的作用。敲低MAFG-AS1可抑制JAK2/STAT3信号通路的活性。此外，敲低MAFG-AS1可减少肿瘤生长。
结论
敲低MAFG-AS1可通过MAFG-AS1/miR-3196/TFAP2A调控轴激活JAK2/STAT3信号通路，从而减弱乳腺癌进展。
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