Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway

Abstract

The transforming growth factor-β (TGF-β) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-β signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-β transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3′-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-β to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-β signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4.

Competition between human cells by entosis

“…For BrdU staining, additional procedures of denaturing with 2N HCl for 20 min and quenching with 0.1 M Na 2 B 4 O 7 for 2 min were performed before blocking. Immunoblotting was performed as previously described [46].…”

Induction of entosis by epithelial cadherin expression

“…To examine transformed growth [46], cells (5 000 cells) were embedded into growth media + 0.4% low melting agarose (Sigma), and plated onto preformed growth media + 0.5% agarose pads in 6-well plates. 1 ml of full media with or without Y27632 was added after agarose was solidified at room temperature.…”

Upregulated microRNA-199a-5p inhibits nuclear receptor corepressor 1 translation in mice with non-alcoholic steatohepatitis

“…Thus, miR-122, a key regulator of cholesterol and fatty-acid metabolism, may be an attractive therapeutic target for metabolic diseases, including NASH. miR-199 has been reported to affect the proliferation and apoptosis of hepatoma carcinoma cells (38), and regulates the TGF-β signaling pathway, by directly targeting Smad4 (39). Furthermore, Murakami et al (16) have shown that overexpression of miR-199 is associated with the progression of liver fibrosis.…”