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. Liver carcinoma cell line Huh7 samples were transfected with each siRNA (siCtrl #2 scramble, #3 scramble, siELIT-1 #2, or siELIT-1 #3). At 24 h after the first transfection, cells were transfected with these siRNAs, again. At 6 h after the second transfection, cells were treated with or without 10 ng/mL TGF-b for 48 h. Total RNA was isolated from cultured cells using RNeasy Mini Kit and followed by microarray analysis.