miR-322-5p targets IGF-1 and is suppressed in the heart of rats with pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is characterised by remodelling of the pulmonary vasculature leading to right ventricular hypertrophy. Here, we show that miR-322-5p (the rodent orthologue of miR-424-5p) expression is decreased in the right ventricle of monocrotaline-treated rats, a model of PAH, whereas a putative target insulin-like growth factor 1 (IGF-1) is increased. IGF-1 mRNA was enriched 16-fold in RNA immunoprecipitated with Ago2, indicating binding to miR-322-5p. In cell transfection experiments, miR-322-5p suppressed the activity of a luciferase reporter containing a section of the IGF-1 3' untranslated region (UTR) as well as IGF-1 mRNA and protein levels. Taken together, these data suggest that miR-322 targets IGF-1, a process downregulated in PAH-related RV hypertrophy.

Keywords: IGF‐1; miR‐322/424; pulmonary arterial hypertension; right ventricular hypertrophy.

Figure 1

(A) Pulmonary arterial pressure measured as mm of mercury (Hg) in five monocrotaline (MCT)‐treated and seven PBS‐treated rats. (B) Right ventricle weight normalised to left ventricle and septum weight in nine MCT‐treated and eight PBS‐treated rats. (C) Normalised miR‐322‐5p expression in MCT‐ and PBS‐treated rats. D. IGF‐1 expression levels in MCT‐ and PBS‐treated rats.

Figure 2

(A) (top) miR‐322‐5p mature sequence showing putative bind sites to IGF‐1 3′UTR with complimentary bases bolded and seed sequence match site underlined; (bottom) miR‐424‐5p mature sequence showing multiple bind sites to IGF‐1 coding region and 3′UTR with complimentary bases bolded and seed sequence match site underlined. (B) qPCR‐measured enrichment of mRNA from immunoprecipitation of argonaute 2 (Ago2) protein in cells transfected with miR‐322‐5p compared to control miRNA‐transfected cells. (C) Expression of pMIR‐REPORT luciferase with IGF‐1 3′UTR sequence in cells transfected with miR‐322‐5p compared to scrambled controls.

Figure 3

(A) IGF‐1 expression in C2C12 mouse myoblasts treated with miR‐322‐5p, miR‐322‐5p + antagomir inhibitor, or control miRNA. (B) IGF‐1 expression in LHCN‐M2 human myoblasts treated with miR‐424‐5p, miR‐424‐5p + antagomir inhibitor, or control miRNA. (C) Secreted IGF‐1 protein levels normalised to total secreted protein in LHCN‐M2 human myoblasts transfected with miR‐424‐5p or control miRNA. (D) Western blot showing protein levels of IGF‐1 in cells transfected with miR‐424‐5p (left) and control miRNA (right). Ponceau S staining of membrane shown below.