MicroRNA-140-5p and SMURF1 regulate pulmonary arterial hypertension
Alexander M.K. Rothman, … , David J. Rowlands, Allan Lawrie
Alexander M.K. Rothman, … , David J. Rowlands, Allan Lawrie
Published May 23, 2016
Citation Information: J Clin Invest. 2016;126(7):2495-2508. https://doi.org/10.1172/JCI83361.
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Research Article Pulmonology

MicroRNA-140-5p and SMURF1 regulate pulmonary arterial hypertension

Abstract

Loss of the growth-suppressive effects of bone morphogenetic protein (BMP) signaling has been demonstrated to promote pulmonary arterial endothelial cell dysfunction and induce pulmonary arterial smooth muscle cell (PASMC) proliferation, leading to the development of pulmonary arterial hypertension (PAH). MicroRNAs (miRs) mediate higher order regulation of cellular function through coordinated modulation of mRNA targets; however, miR expression is altered by disease development and drug therapy. Here, we examined treatment-naive patients and experimental models of PAH and identified a reduction in the levels of miR-140-5p. Inhibition of miR-140-5p promoted PASMC proliferation and migration in vitro. In rat models of PAH, nebulized delivery of miR-140-5p mimic prevented the development of PAH and attenuated the progression of established PAH. Network and pathway analysis identified SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) as a key miR-140-5p target and regulator of BMP signaling. Evaluation of human tissue revealed that SMURF1 is increased in patients with PAH. miR-140-5p mimic or SMURF1 knockdown in PASMCs altered BMP signaling, further supporting these factors as regulators of BMP signaling. Finally, Smurf1 deletion protected mice from PAH, demonstrating a critical role in disease development. Together, these studies identify both miR-140-5p and SMURF1 as key regulators of disease pathology and as potential therapeutic targets for the treatment of PAH.

Authors

Alexander M.K. Rothman, Nadine D. Arnold, Josephine A. Pickworth, James Iremonger, Loredana Ciuclan, Robert M.H. Allen, Sabine Guth-Gundel, Mark Southwood, Nicholas W. Morrell, Matthew Thomas, Sheila E. Francis, David J. Rowlands, Allan Lawrie

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Figure 6

miR-140-5p regulates key mediators of PAH pathology.

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miR-140-5p regulates key mediators of PAH pathology. (A) Predicted PH-re...
(A) Predicted PH-related direct targets of miR-140-5p. (B and C) qPCR (B) and protein expression by Western blot (C) showing expression of miR-140-5p targets ALK5, PDGFRA, and SMURF1 in PASMCs at 72 hours following transfection with miR-140-5p mimic (M), miR-140-5p inhibitor (I), or scramble control (S) (B: n = 6, C: n = 5, B and C: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA with Tukey’s post-test correction, mean ± SEM). (D) Whole lung levels of SMURF1 protein correlate inversely with miR-140-5p (n = 38, P < 0.05, r = –0.36, Spearman rank). (E) Increased whole blood levels of SMURF1 mRNA were observed in patients with IPAH when compared with HV (IPAH n = 20, HV n = 16, *P < 0.05, unpaired 2-tailed Student’s t test, mean ± SEM). P, prevention; T, therapeutic. (F) Representative photomicrographs demonstrating immunoreactivity of SMURF1 in endothelial and SMCs of pulmonary vascular lesions in the transplanted lungs of patients with BMPR2 mutation–associated heritable PAH, associated PAH, and non-PAH control lung (photomicrographs representative of n = 26: APAH n = 8, IPAH n = 8, FPAH, n = 6, control n = 4). Original magnification, ×200 (hereditary PAH and control); ×400 (associated PAH). Scale bars: 50 μm. (G) Chromosomal location of putative miR-140-5p–binding site in the 3′ UTR of SMURF1. (H and I) Transfection of PASMCs with miR-140-5p inhibitor (H) and mimic (I) alter luciferase activity of SMURF1 3′ UTR construct at 24 hours (H and I: n = 4, **P < 0.01, ****P < 0.0001, 2-tailed Mann-Whitney U test, mean ± SEM).