Versican 3'-非翻译区(3'-UTR)作为非编码竞争内源性RNA(ceRNA)通过调控MicroRNAs的功能在乳腺癌中的作用

The Roles of Versican 3'-Untranslated Region (3'-UTR) Functioning as The Non-coding Competing Endogenous RNA (ceRNA) by Regulating MicroRNAs Activity in Breast Carcinoma Nowadays in life sciences, it is becoming another focus area that study on the interaction mechanism in non-coding RNA functions in regulating cell activities. In our previous studies, we found that the non-coding 3'untranslated region (UTR) plays an important role in the regulation of endogenous miRNA functions (Rutnam ZJ, Nature Communications 5:2914, 1-15, 2014; Jeyapalan et al., Nucleic Acids Research 39(8):3026-41, 2010; Fang L and Du WW et al., FASEB J. 27(3), 907-19, 2013). On the other hand, we reported that Versican can play important roles in cell apoptosis (LaPierre et al., Cancer Research, 67, 4742-4750, 2007). Based on the above results, we will investigate the roles and mechanism of Versican 3'-untranslated region (3'-UTR) in regulating cell activities by functioning as the non-coding competitive endogenous RNA (ceRNA), which can bind and regulate endogenous miRNA functions, thus modulating its potential targets. In addition, we will use breast carcinoma cell lines stably transfected with Versican 3'-UTR to examine its effects on tumor cell proliferation, survival, apoptosis, migration, invasion, colony formation, and endothelial cell activities. We will also design Versican 3'-UTR siRNAs to confirm the functions of Versican 3'-UTR. Furthermore, we will use transgenic mice expressing Versican 3'-UTR and clinical breast carcinoma patients samples to study whether or not expression of this 3'-UTR promotes breast carcinoma growth by regulating expression of Versican isoforms. Finally, in our preliminary results we found that 4T1 cells transfected with Versican 3'-UTR showed reduction of miR-199a-3p compared with cells transfected with the control vector. And in the 4T1 cell line expression of Versican 3'-UTR reduced breast carcinoma cells proliferation. Therefore, we believe that this project will provide important new views and strategies on the pathogenesis, diagnosis and treatment of breast carcinoma.

研究非编码RNA之间的相互调节机制从而调控生命体已成为又一新兴研究热点。我们的研究表明，非编码3'-UTR能够调节内源性miRNA的功能(Nature Communications; Nucleic Acids Research; FASEB J.)；且Versican在细胞凋亡中起着重要作用(Cancer Research)。基于此本课题将继续研究乳腺癌中Versican 3'-UTR作为非编码竞争内源性RNA(ceRNA)调控miRNA 及其相关靶蛋白的作用及作用机制；研究Versican 3'-UTR对乳腺癌细胞的调控作用，并用siRNAs确认其功能；研究其转基因小鼠和乳腺癌标本中Versican亚型的表达，从而研究其对乳腺癌的作用。初步结果显示Versican 3'-UTR结合miR-199a-3p，并抑制了乳腺癌细胞增殖。本课题将为乳腺癌的发生机制和诊治提供重要理论依据及新策略。

非编码RNA之间的相互调节机制，从而调控生物体已成为生命科学领域的又一新兴研究热点。我们发现CD44 3'-UTR能够调节内源性miRNA的功能（Nucleic Acids Research）。基于此本课题将研究Versican 3'-UTR对乳腺癌细胞的调控作用。我们的研究结果发现Versican 3'-UTR能够抑制乳腺癌细胞增殖和细胞克隆的形成，并抑制了G2/M期的乳腺癌细胞。体内小鼠的荷瘤实验结果表明与对照组相比Versican 3'-UTR能够显著抑制肿瘤生长。肿瘤标志物免疫组化染色结果显示与对照组相比Ki67和CD31的表达显著降低。进而，我们研究了Versican亚型的表达对乳腺癌生长的作用。我们发现Versican 3'-UTR主要促进了Versican V3亚型的表达，表明V3亚型在乳腺癌的发展中发挥着重要作用。. 此外我们研究Versican 3'-UTR与靶蛋白之间的调节关系，结果表明Versican 3'-UTR与靶蛋白Rb1间存在相互作用机制。Versican 3'-UTR能够促进Rb1蛋白的表达。我们利用siRNA确认Versican 3'-UTR对靶蛋白的调控作用。结果表明Versican 3'-UTR siRNA能够明显抑制Rb1蛋白的表达。. 最后我们研究了Versican 3'-UTR作为ceRNA调控miRNA的功能，并证实miRNA能够靶向3'-UTR。结果表明，Versican 3'-UTR与miR-199a和miR-144存在结合位点。Versican 3'-UTR通过调控miR-199a和miR-144进而促进了Rb1蛋白的表达。. 综上，通过上述研究我们发现Versican 3'-UTR作为ceRNA通过调控miR-199a和miR-144进而促进了Rb1蛋白的表达，从而影响乳腺癌的发生发展。本课题将为乳腺癌的发生机制和诊治提供重要理论依据及新策略。