Copy number gain of ZEB1 mediates a double-negative feedback loop with miR-33a-5p that regulates EMT and bone metastasis of prostate cancer dependent on TGF-β signaling
Copy number gain of ZEB1 mediates a double-negative feedback loop with miR-33a-5p that regulates EMT and bone metastasis of prostate cancer dependent on TGF-β signaling
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DOI：
10.7150/thno.36735
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发表时间：
2019-01-01
期刊：
影响因子：
12.4
通讯作者：
Ren, Dong
中科院分区：
文献类型：
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作者：
Dai, Yuhu;Wu, Zhengquan;Ren, Dong
背景资料：ZEB 1和miRNA之间的相互抑制环已被广泛报道在各种人类肿瘤类型的肿瘤进展和转移中起重要作用。本研究旨在探讨ZEB 1与miR-33 a-5 p之间的双负反馈环在前列腺癌骨转移中的作用及其机制。进行统计学分析以评估PCa患者中miR-33 a-5 p表达与临床病理特征以及总体和无骨转移生存期之间的临床相关性。通过体外EMT和Transwell实验以及体内左心室接种模型研究miR-33 a-5 p在PCa骨转移中的生物学作用。利用siRNA文库、实时荧光定量PCR和染色质免疫沉淀（ChIP）技术研究PCa中miR-33 a-5 p表达降低的机制。采用生物信息学分析、Western blotting和荧光素酶报告基因分析等方法研究miR-33 a-5 p与其潜在靶点的关系。结果：miR-33 a-5 p在有骨转移的PCa组织和骨源性细胞中表达下调，miR-33 a-5 p的低表达与PCa患者的晚期临床病理特征、总生存期和无骨转移生存期较短密切相关。上调miR-33 a-5 p可抑制PCa细胞的EMT、侵袭和迁移，而沉默miR-33 a-5 p可促进PCa细胞的EMT、侵袭和迁移。重要的是，上调miR-33 a-5 p在体内显著抑制PC-3细胞的骨转移。我们的研究结果进一步揭示了由拷贝数增加诱导的反复ZEB 1上调在转录上抑制了miR-33 a-5 p的表达，导致了骨转移性PCa组织中miR-33 a-5 p的表达降低。反过来，miR-33 a-5 p与ZEB 1以靶点非依赖性方式形成双负反馈环，这依赖于TGF-β信号传导。结论：ZEB 1启动的TGF-β信号依赖性miR-33 a-5 p负反馈环与PCa骨转移密切相关。
Background: The reciprocal repressive loop between ZEB1 and miRNAs has been extensively reported to play an important role in tumor progression and metastasis of various human tumor types. The aim of this study was to elucidate the role and the underlying mechanism of the double-negative feedback loop between ZEB1and miR-33a-5p in bone metastasis of prostate cancer (PCa).Methods: miR-33a-5p expression was examined in 40 bone metastatic and 165 non-bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-33a-5p expression and clinicopathological characteristics, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-33a-5p in bone metastasis of PCa were investigated both by EMT and the Transwell assay in vitro, and by a mouse model of left cardiac ventricle inoculation in vivo. siRNA library, real-time PCR and chromatin immunoprecipitation (ChIP) were used to identify the underlying mechanism responsible for the decreased expression of miR-33a-5p in PCa. Bioinformatics analysis, Western blotting and luciferase reporter analysis were employed to examine the relationship between miR-33a-5p and its potential targets. Clinical correlation of miR-33a-5p with its targets was examined in human PCa tissues and primary PCa cells.Results: miR-33a-5p expression was downregulated in PCa tissues with bone metastasis and bone-derived cells, and low expression of miR-33a-5p strongly and positively correlated with advanced clinicopathological characteristics, and shorter overall and bone metastasis-free survival in PCa patients. Upregulating miR-33a-5p inhibited, while silencing miR-33a-5p promoted EMT, invasion and migration of PCa cells. Importantly, upregulating miR-33a-5p significantly repressed bone metastasis of PC-3 cells in vivo. Our results further revealed that recurrent ZEB1 upregulation induced by copy number gains transcriptionally inhibited miR-33a-5p expression, contributing to the reduced expression of miR-33a-5p in bone metastatic PCa tissues. In turn, miR-33a-5p formed a double negative feedback loop with ZEB1 in target-independent manner, which was dependent on TGF-beta signaling. Finally, the clinical negative correlations of miR-33a-5p with ZEB1 expression and TGF-beta signaling activity were demonstrated in PCa tissues and primary PCa cells.Conclusion: Our findings elucidated that copy number gains of ZEB1-triggered a TGF-beta signaling-dependent miR-33a-5p-mediated negative feedback loop was highly relevant to the bone metastasis of PCa.