title: 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.
authors: State Key Laboratory of Proteomics, Genetic Laboratory of Development and Disease, Institute of Biotechnology, 20 Dongdajie, Fengtai District, Beijing 100071, China.
authors: Yan
authors: Y
authors: Zhang
authors: Kai-Ji
authors: KJ
authors: Fan
authors: Qiang
authors: Q
authors: Sun
authors: Ai-Zhong
authors: AZ
authors: Chen
authors: Wen-Long
authors: WL
authors: Shen
authors: Zhi-Hu
authors: ZH
authors: Zhao
authors: Xiao-Fei
authors: XF
authors: Zheng
authors: Xiao
authors: X
authors: Yang
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D041681:NIH 3T3 Cells; D016415:Sequence Alignment; D015398:Signal Transduction; D051901:Smad4 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors*
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; C531408:mirn199 microRNA, human