Exosomal miR-211 contributes to pulmonary hypertension via attenuating CaMK1/PPAR-γaxis

Abstract

Aim: Exsomes play a significant role in increasing pathophysiological processes by delivering their content. Recently, a variety of studies have showed exosomal microRNAs (miRNAs) are involved in pulmonary hypertension (PH) notably. In this study, we found that exosomal miR-211 was overexpressed in hypoxia-induced PH rats but its intrinsic regulation was unclear. Therefore, our aim was to reveal the underlying mechanism which overexpressed exosomal miR-211 targeted in the development of PH.

Methods: 18 male SD rats were randomly divided into normoxia and hypoxia group, housed in normal or hypoxic chamber for 3 weeks respectively. Then, mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance(PVR), right ventricular hypertrophy index(RV/(LV + S)), the percentage of medial wall area (WA%) and the percentage of medial wall thickness (WT%) were measured. Expression of miR-211 in exosomes was detected by qRT-PCR. Expression of Ca²⁺/calmodulin-dependent kinase1(CaMK1)and peroxisome proliferator-activated receptors-γ(PPAR-γ)in lung tissue were detected by Western blot(WB); After miR-211 overexpressed exosomes were injected to rats through caudal vein, mPAP, PVR, RV/(LV + S), WA% and WT% were also measured. Sequentially, hypoxia rats were injected with lentivirus riched in miR-211 inhibitor via tail vein, and PH-related indicators were measured. In vitro, after miR-211 was positively or negatively regulated in pulmonary arterial smooth muscle cell (PASMC) by plasmid transfection, proliferation of PASMC was detected by CCK8, as well as the expression of CaMK1 and PPAR- γ. Further, the relationship between CaMK1 and miR-211 was verified by Dual-Luciferase assay. And the regulatory relationship of CaMK1/PPAR- γ aixs was demonstrated in PASMC.

Results: Evident increases of mPAP, PVR, RVHI, WT% and WA% were observed with hypoxia administration. And the concentration of plasma exosomes in hypoxia rats was increased and positively correlated with the above indexes. miR-211 in exosomes of PH was upregulated while the expression of CaMK1 and PPAR-γ decreased in lung tissues. Further, injection of exosomes overexpressed with miR-211 demonstrated that exosomal miR-211 aggravated PH while inhibition of miR-211 attenuated PH in rats. In vitro, overexpression of miR-211 promoted the proliferation of PASMC and inhibited expression of CaMK1 and PPAR-γ in PASMC. And Dual-luciferase assay demonstrated that CaMK1 was a downstream gene of miR-211. Plasmid transfection experiments indicated that CaMK1 can promote PPAR-γ expression.

Conclusion: Exosomal miR-211 promoted PH via inhibiting CaMK1/PPAR-γ axis, promoting PASMC proliferation in rats.

Keywords: CaMK1; Exosome; PPAR-γ; Pulmonary hypertension; miR-211.