Systems-level regulation of microRNA networks by miR-130/301 promotes pulmonary hypertension
Thomas Bertero, … , Katherine A. Cottrill, Stephen Y. Chan
Thomas Bertero, … , Katherine A. Cottrill, Stephen Y. Chan
Published June 24, 2014
Citation Information: J Clin Invest. 2014;124(8):3514-3528. https://doi.org/10.1172/JCI74773.
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Research Article Pulmonology

Systems-level regulation of microRNA networks by miR-130/301 promotes pulmonary hypertension

Abstract

Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPARγ with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.

Authors

Thomas Bertero, Yu Lu, Sofia Annis, Andrew Hale, Balkrishen Bhat, Rajan Saggar, Rajeev Saggar, W. Dean Wallace, David J. Ross, Sara O. Vargas, Brian B. Graham, Rahul Kumar, Stephen M. Black, Sohrab Fratz, Jeffrey R. Fineman, James D. West, Kathleen J. Haley, Aaron B. Waxman, B. Nelson Chau, Katherine A. Cottrill, Stephen Y. Chan

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Figure 6

The miR-130/301 family represses PPARγ in order to control proliferation in PASMCs via the STAT3-miR-204 regulatory axis.

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The miR-130/301 family represses PPARγ in order to control proliferation...
In normoxia (21% O2) or hypoxia (0.2% O2, 24 hours), either forced expression of miR-130a mimic versus control (NC) or inhibition of miR-130a (anti-miR-130a) versus inhibition of the entire miR-130/301 family (tiny-LNA-130) versus control (NC) was performed in cultured PASMCs (A–D). Immunoblotting (A), gel densitometry (B), and RT-qPCR (C) demonstrated that forced miR-130a expression decreased PPARγ, accompanied by an increase in STAT3 and activated phosphorylated STAT3 (P-STAT3; A and B), as well as decreased miR-204 (C). Importantly, as in PAECs, inhibition of the entire miR-130/301 family in PASMCs reversed such downstream gene/subordinate miRNA alterations to a greater extent than inhibition of miR-130a alone (A–D), thus demonstrating the importance of the coordinated actions of this miRNA family. (D) As assessed by BrdU incorporation in exponentially growing PASMCs, proliferation was augmented during forced expression of miR-130a mimics but decreased during expression of miR-204. MiR-130a–induced proliferation was reversed when miR-204 was simultaneously expressed, thus confirming the direct dependence of miR-130a on miR-204 in this cell type. (E) In PASMCs, we have validated that the miR-130/301-PPARγ regulatory axis controls proliferation by increasing STAT3 expression and activity and repressing subordinate miR-204 expression. In C, for each miRNA, mean expression in control groups (miR-NC or anti-miR-NC) was assigned a fold change of 1, to which relevant samples were compared. In all panels, data are expressed as mean ± SD (*P < 0.05; **P < 0.01).