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A2236 - Downregulation of Cleavage Factor 25 by TGFb1 Contributes to Pulmonary Fibrosis Through Alternative Polyadenylation of mRNAs
Author Block: J. Ko1, T. W. Mills1, T. Mertens1, N. Chen1, J. Huang2, F. Luo1, J. Molina1, K. Volcik1, Y. Zhou3, M. R. Blackburn1; 1Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, TX, United States, 2Department of Geriatrics, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China, 3Brown University, Providence, RI, United States.
Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease characterized by differentiation of fibroblasts to myofibroblasts and accumulation of extracellular metric (ECM) in interstitial tissues leading to hypoxia in the patients. Myofibroblast is a key player in the ECM deposition and IPF development in humans. However, the mechanism for ECM production by myofibroblasts is not completely understood. Alternative polyadenylation (APA) is a cellular process that controls the length of 3’-untranslated regions(3’UTR) of mRNAs. It is known that 3’UTR shortening of mRNAs by APA leads to robust protein production, due to inefficient miRNA binding to the mRNAs in cancer. Moreover, it was reported that deletion of cleavage factor 25, a subunit in cleavage and polyadenylation machinery, promotes 3’UTR shortening of mRNAs. However, it is not known whether APA of mRNAs by CFIm25 reduction contributes to pulmonary fibrosis by targeting profibrotic and matrix genes. This presentation will discuss whether 1) CFIm25 reduction contributes to pulmonary fibrosis and 2) what factor promotes CFIm25 reduction.
Methods: Lung samples from IPF patients or healthy donors were used to examine expression of CFIm25 in the lungs by western blot. Immunohistochemistry was conducted in IPF or healthy lungs to find cells which exhibit downregulated CFIm25. For mechanistic study, primary human lung fibroblasts were isolated from lungs of healthy donors or IPF patients and were cultured. Proteins, mRNA, and APA usage were evaluated using western blot, RT-qPCR, and RNA-seq.
Results: CFIm25 was significantly downregulated in fibroblasts in the lungs of IPF patients. CFIm25 knockdown by siRNA promoted collagen and fibronectin expression in healthy primary human lung fibroblasts. Upon bleomycin treatment, CFIm25 knockout mice, which have deleted CFIm25 in fibroblasts, developed worsened pulmonary fibrosis, suggesting that CFIm25 deletion exacerbates experimental lung fibrosis in mice. RNA-seq data revealed that CFIm25 knockdown by siRNA induces global mRNA shortening through APA in transcriptome and the shortened APA genes were enriched for profibrotic pathways, such as Wnt5 and transforming growth factor beta 1 (TGFβ1) pathways. Through a screening, it was found that TGFβ1 downregulates CFIm25 in human lung fibroblasts at protein and mRNA levels. TGFβ1 made CFIm25 mRNAs unstable through upregulation of miR203, which targets 3’UTR of CFIm25. Phosphorylated Smad3, a downstream of TGFβ1, worked as a transcription factor for miR203 upregulation following TGFβ1 treatment.
Conclusion: Downregulation of cleavage factor 25 by TGFβ1 contributes to pulmonary fibrosis through alternative polyadenylation of mRNAs.