Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation
Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn
Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn
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Research Article Pulmonology

Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

Abstract

Idiopathic pulmonary fibrosis (IPF) is a deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) is a key factor that drives the disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors through the alteration of miRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrated that CFIm25, a global regulator of APA, was downregulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in α–smooth muscle actin–positive (α-SMA–positive) fibroblasts. Following CFIm25 knockdown in healthy human lung fibroblasts, we identified 808 genes with shortened 3′-UTRs, including those involved in the TGF-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key profibrotic factors was suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrated that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhanced pulmonary fibrosis after bleomycin exposure. Collectively, our results identified CFIm25 downregulation as an important mechanism for elevating profibrotic gene expression in pulmonary fibrosis.

Authors

Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn

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Figure 2

RNA-Seq identifies CFIm25 target genes involved in important fibrotic and cancer pathways.

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RNA-Seq identifies CFIm25 target genes involved in important fibrotic an...
(A) Western blot analysis of CFIm25, COL1, and FN expression in healthy human fibroblasts (CCD8-Lu) transfected with control siRNA (si-Con) or siRNAs targeting CFIm25 (si-CFIm25). GAPDH was used as a loading control. (B) Diagram shows pPAS and dPAS, and 2 sets of primers designed to target the translated region (P1) and distal region (P2) of the 3′-UTR. qRT-PCR was performed to demonstrate COL1A1 and FN1 transcript expression (lower left panel) and dPAS usage (lower right panel) after knockdown of CFIm25 using 2 different siRNAs (no. 1 and no. 2). Results in the lower left panel are shown as log2 (fold changes vs. control siRNA–transfected samples) ± SEM (n = 3 biological replicates), and results in the lower right panel are shown as the log2 ratio of (percentage of long transcript in si-CFIm25/percentage of long transcript in si-Con). *P < 0.05, by 1-sample t test versus 0. (C) Scatterplot of percentage PDUIs in control and CFIm25-knockdown cells, in which mRNAs were significantly shortened (n = 808) or lengthened (n = 29) after CFIm25 knockdown in CCD8-Lu cells. (D) Functional annotation assay of CFIm25 targets. (E) RNA-Seq read density for 3′-UTR, terminal exon, and upstream exon(s) of VMA21 in control or CFIm25 siRNA–transfected CCD8-Lu cells. Numbers on the x axis indicate the RNA-Seq read coverage. (F) qRT-PCR was performed to demonstrate dPAS usage of VMA21. n = 3. *P < 0.01, by 1-sample t test versus 0. (G) Western blotting was used to verify VMA21 expression after CFIm25 knockdown in CCD8-Lu cells. KD, knockdown.