Long noncoding RNA ELIT-1 acts as a Smad3 cofactor to facilitate TGF-β/Smad signaling and promote epithelial-mesenchymal transition

TGF-β is involved in various biological processes, including development, differentiation, growth regulation, and epithelial-mesenchymal transition (EMT). In TGF-β/Smad signaling, receptor-activated Smad complexes activate or repress their target gene promoters. Smad cofactors are a group of Smad-binding proteins that promote recruitment of Smad complexes to these promoters. Long noncoding RNAs (lncRNAs), that behave as Smad cofactors have thus far not been identified. Here, we characterize a novel lncRNA EMT-associated lncRNA induced by TGF-β-1(ELIT-1). ELIT-1 was induced by TGF-β-stimulation via the TGF-β/Smad pathway in TGF-β-responsive cell lines. ELIT-1-depletion abrogated TGF-β-mediated EMT progression and expression of TGF-β target genes including Snail, a transcription factor critical for EMT. A positive correlation between high expression of ELIT-1 and poor prognosis in lung adenocarcinoma and gastric cancer patients suggests that ELIT-1 may be useful as a prognostic and therapeutic target. RIP assays revealed that ELIT-1 bound to Smad3, but not Smad2. In conjunction with Smad3, ELIT-1 enhanced Smad-responsive promoter activities by recruiting Smad3 to the promoters of its target genes including Snail, other TGF-β-target genes, and ELIT-1 itself. Collectively, these data show that ELIT-1 is a novel trans-acting lncRNA that forms a positive feedback loop to enhance TGF-β/Smad3 signaling and promote EMT progression. To identify the genes induced by HBV replication, HBV genome containing episomal vectors (Ae, Bj35, Bj56) or its empty vector (pEB) were transfected in liver carcinoma cell line Huh7. After puromycin screening, the transfectants were subjected to purify its total RNA and followed by microarray analysis.

转化生长因子β（TGF-β）参与诸多生物学过程，包括发育、分化、生长调控以及上皮间质转化（epithelial-mesenchymal transition, EMT）。在TGF-β/Smad信号通路中，受体激活型Smad复合物可激活或抑制其靶基因的启动子。Smad辅因子是一类能够促进Smad复合物招募至这些启动子的Smad结合蛋白，目前尚未发现可作为Smad辅因子的长链非编码RNA（long noncoding RNAs, lncRNAs）。本研究对一种由TGF-β1诱导的新型长链非编码RNA——EMT相关lncRNA（EMT-associated lncRNA induced by TGF-β1, ELIT-1）进行了表征：ELIT-1可在TGF-β应答细胞系中通过TGF-β/Smad通路被TGF-β刺激诱导表达；敲低ELIT-1会阻断TGF-β介导的EMT进程以及包括Snail在内的TGF-β靶基因的表达，而Snail是调控EMT的关键转录因子。ELIT-1高表达与肺腺癌和胃癌患者的不良预后呈正相关，提示ELIT-1可作为预后评估与治疗靶点。RNA免疫沉淀（RNA immunoprecipitation, RIP）实验结果显示，ELIT-1可结合Smad3，但无法结合Smad2；ELIT-1可与Smad3协同，通过将Smad3招募至包括Snail在内的靶基因、其他TGF-β靶基因以及ELIT-1自身的启动子区域，增强Smad应答启动子的活性。综上，本研究数据表明ELIT-1是一种新型反式作用长链非编码RNA，可通过形成正反馈环路增强TGF-β/Smad3信号通路并促进EMT进程。为鉴定HBV复制诱导的基因，研究人员将携带HBV基因组的游离型载体（Ae、Bj35、Bj56）及其空载体（pEB）分别转染至肝癌细胞系Huh7中；经嘌呤霉素筛选后，收集转染细胞的总RNA并进行微阵列分析。