Effect of miRNA-381-3p/MuRF1 axis on cardiopulmonary injury in mice with hypoxic pulmonary hypertension

doi:10.11958/20251127

Abstract

Abstract:

Objective To explore the effect of microRNA-381-3p (miR-381-3p) /MuRF1 axis on cardiopulmonary injury in hypoxia-induced pulmonary hypertension (HPH) mice and its potential mechanisms. Methods Sixty mice were randomly assigned to four groups: the normal control group (NC), the hypobaric hypoxia-induced pulmonary hypertension (HPH) group, the HPH + agomir control group and the HPH + miR-381-3p agomir analog group (HPH + miR-381-3p agomir), with 15 mice in each group. The HPH mouse model was established using a low-pressure and hypoxic artificial chamber. Three weeks prior to the establishment of the HPH model, miR-381-3p agomir and its corresponding control agomir were prepared by dissolving them in RNA-free phosphate-buffered saline (PBS) according to the experimental requirements. These solutions were administered via tail vein injection at a dose of 10 mg/kg, twice weekly for three consecutive weeks. Right heart function was assessed using echocardiography. Right ventricular systolic pressure (RVSP) was measured via cardiac catheterization. Pulmonary vascular remodeling was evaluated through hematoxylin and eosin (HE) staining. Levels of inflammatory cytokines in bronchoalveolar lavage fluid were quantified using enzyme-linked immunosorbent assay (ELISA). Real-time quantitative fluorescent PCR (RT-qPCR) was employed to analyze the mRNA expression levels of miR-381-3p and MuRF1. Potential targets of miR-381-3p were predicted, and pathway enrichment analysis was conducted. A dual-luciferase reporter gene assay was performed to confirm the direct regulatory effect of miR-381-3p on MuRF1. Results Compared with the NC group, the mRNA expression of miR-381-3p was significantly decreased in both the HPH group and the HPH+agomir control group, whereas the mRNA expression of MuRF1 was significantly increased (P<0.05). In contrast, compared with the HPH group and the HPH+agomir control group, the mRNA expression of miR-381-3p was significantly increased in the HPH+miR-381-3p agomir group, while the mRNA expression of MuRF1 was significantly decreased (P<0.05). Additionally, compared with the NC group, RVSP, right ventricular anterior wall thickness (RVAW), right ventricular hypertrophy index (RVHI), right ventricular collagen volume fraction (CVF), distal pulmonary artery wall thickness ratio (WT), pulmonary artery wall area ratio (WA), as well as IL-1β, IL-6 and TNF-α levels in alveolar lavage fluid were significantly increased in the HPH group and the HPH+agomir control group, whereas the right ventricular diameter (RVID) was significantly decreased (P<0.05). Conversely, compared with the HPH group and the HPH+agomir control group, RVSP, RVAW, RVHI, right ventricular CVF, WT, Wa and RVID were decreased in the HPH+miR-381-3p agomir group, and IL-1β, IL-6, and TNF-α levels of alveolar lavage fluid were significantly decreased (P<0.05). Furthermore, the downstream target genes of miR-381-3p were predicted in the database, and MuRF1 was a potential target, and the Cytoskeleton in muscle cells ranked first in the significant enrichment of target genes. Compared with WT-MuRF1+mimic control group, the luciferase activity was decreased in the WT-MuRF1+miR-381-3p mimic group (P<0.05). There was no significant difference in the luciferase activity between the Mut-MuRF1+mimic control group and the Mut-MuRF1+miR-381-3p mimic group. Conclusion Overexpression of miR-381-3p can improve cardiopulmonary injury in HPH mice, and the mechanism may be related to the targeted inhibition of MuRF1 by miR-381-3p.

Key words: hypoxia, hypertension, pulmonary, muscle-specific ring finger protein 1, microRNA-381-3p, myocardial fibrosis, pulmonary artery remodeling

CLC Number:

R544.16，R541.3

Figures/Tables 9

References 22

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基因名称	引物序列（5'→3'）	产物大小/bp
miR-381-3p	上游：CGCGTATACAAGGGCAAGCT	75
miR-381-3p	下游：AGTGCAGGGTCCGAGGTATT	75
U6	上游：ATTGGAACGATACAGAGAAGATT	95
U6	下游：GGAACGCTTCACGAATTTG	95
MuRF1	上游：GTGTGAGGTGCCTACTTGCTC	201
MuRF1	下游：GCTCAGTCTTCTGTCCTTGGA	201
β-actin	上游：CATGTACGTTGCTATCCAGGC	181
β-actin	下游：CTCCTTAATGTCACGCACGAT	181

基因名称	引物序列（5'→3'）	产物大小/bp
miR-381-3p	上游：CGCGTATACAAGGGCAAGCT	75
miR-381-3p	下游：AGTGCAGGGTCCGAGGTATT	75
U6	上游：ATTGGAACGATACAGAGAAGATT	95
U6	下游：GGAACGCTTCACGAATTTG	95
MuRF1	上游：GTGTGAGGTGCCTACTTGCTC	201
MuRF1	下游：GCTCAGTCTTCTGTCCTTGGA	201
β-actin	上游：CATGTACGTTGCTATCCAGGC	181
β-actin	下游：CTCCTTAATGTCACGCACGAT	181

组别	RVSP/mmHg	RVID/mm	RVAW/mm	RVHI
NC组	19.17±2.03	2.02±0.18	0.19±0.01	0.18±0.02
HPH组	38.31±2.96^a	1.26±0.09^a	0.39±0.04^a	0.46±0.06^a
HPH+agomir control组	37.01±3.08^a	1.31±0.14^a	0.42±0.07^a	0.49±0.07^a
HPH+miR-381-3p agomir组	28.32±2.93^bc	1.77±0.16^bc	0.33±0.01^bc	0.29±0.03^bc
F	152.700^＊＊	94.420^＊＊	93.360^＊＊	130.800^＊＊

组别	RVSP/mmHg	RVID/mm	RVAW/mm	RVHI
NC组	19.17±2.03	2.02±0.18	0.19±0.01	0.18±0.02
HPH组	38.31±2.96^a	1.26±0.09^a	0.39±0.04^a	0.46±0.06^a
HPH+agomir control组	37.01±3.08^a	1.31±0.14^a	0.42±0.07^a	0.49±0.07^a
HPH+miR-381-3p agomir组	28.32±2.93^bc	1.77±0.16^bc	0.33±0.01^bc	0.29±0.03^bc
F	152.700^＊＊	94.420^＊＊	93.360^＊＊	130.800^＊＊

组别	WT	WA
NC组	22.31±3.18	20.15±3.89
HPH组	45.24±4.08^a	49.02±8.17^a
HPH+agomir control组	47.63±4.79^a	50.12±7.03^a
HPH+miR-381-3p agomir组	30.39±2.73^bc	28.28±4.88^bc
F	153.600^＊＊	87.230^＊＊