miRNA-381-3p/MuRF1轴对低氧性肺动脉高压小鼠心肺损伤的影响

doi:10.11958/20251127

天津医药 ›› 2025, Vol. 53 ›› Issue (6): 571-577.doi: 10.11958/20251127

miRNA-381-3p/MuRF1轴对低氧性肺动脉高压小鼠心肺损伤的影响

吴宾¹(), 杨自更¹, 金玲², 张婧³, 韦红梅³, 蔡冰冰¹, 魏玉英⁴^,^△()

1 新疆军区总医院核医学科（邮编830099）
2 新疆军区总医院老年医学科（邮编830099）
3 新疆军区总医院营养科（邮编830099）
4 新疆军区总医院重症医学科（邮编830099）

收稿日期:2025-03-19 修回日期:2025-04-07 出版日期:2025-06-15 发布日期:2025-06-20
通讯作者: ^△E-mail：gdp19800808@126.com
作者简介:吴宾（1985），男，主治医师，主要从事肺动脉高压的发生发展机制研究。E-mail：xjwubin9210@163.com
基金资助:
新疆维吾尔族自治区自然科学基金面上项目(2022D01C644)

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

WU Bin¹(), YANG Zigeng¹, JIN Ling², ZHANG Jing³, WEI Hongmei³, CAI Bingbing¹, WEI Yuying⁴^,^△()

1 Department of Nuclear Medicine, General Hospital of Xinjiang Military Command, Urumqi 830099, China
2 Department of Geriatrics, General Hospital of Xinjiang Military Command, Urumqi 830099, China
3 Department of Nutrition, General Hospital of Xinjiang Military Command, Urumqi 830099, China
4 Department of Critical Care Medicine, General Hospital of Xinjiang Military Command, Urumqi 830099, China

Received:2025-03-19 Revised:2025-04-07 Published:2025-06-15 Online:2025-06-20
Contact: ^△E-mail: gdp19800808@126.com

吴宾, 杨自更, 金玲, 张婧, 韦红梅, 蔡冰冰, 魏玉英. miRNA-381-3p/MuRF1轴对低氧性肺动脉高压小鼠心肺损伤的影响[J]. 天津医药, 2025, 53(6): 571-577.

WU Bin, YANG Zigeng, JIN Ling, ZHANG Jing, WEI Hongmei, CAI Bingbing, WEI Yuying. Effect of miRNA-381-3p/MuRF1 axis on cardiopulmonary injury in mice with hypoxic pulmonary hypertension[J]. Tianjin Medical Journal, 2025, 53(6): 571-577.

摘要/Abstract

摘要：

目的探讨微小RNA-381-3p（miR-381-3p）/肌肉环指蛋白1（MuRF1）轴在低氧性肺动脉高压（HPH）小鼠心肺损伤中的作用及可能机制。方法将60只C57BL/6小鼠随机分为对照（NC）组、HPH组、HPH+类似物对照（HPH+agomir control）组、HPH+miR-381-3p类似物组（HPH+miR-381-3p agomir）组，每组15只。采用低压低氧人工舱建立HPH小鼠模型，HPH模型构建前3周，按分组要求将miR-381-3p agomir和相当剂量对照agomir control溶解在无RNA酶的PBS中，按照每次10 mg/kg的剂量尾静脉注射，每周2次，持续3周。心脏超声检测小鼠右心功能；心导管测定小鼠右心室收缩压（RVSP）；HE染色评估肺血管重塑；酶联免疫吸附试验（ELISA）测定肺泡灌洗液炎性因子水平；实时荧光定量PCR（RT-qPCR）检测miR-381-3p和MuRF1 mRNA表达；预测miR-381-3p的靶点并进行富集通路分析；双萤光素酶报告基因实验检测miR-381-3p对MuRF1的靶向调控作用。结果与NC组相比，HPH组和HPH+agomir control组miR-381-3p mRNA表达降低，而MuRF1 mRNA表达增高（P<0.05）；与HPH组和HPH+agomir control组相比，HPH+miR-381-3p agomir组miR-381-3p表达增加，而MuRF1 mRNA表达降低（P<0.05）。与NC组相比，HPH组和HPH+agomir control组RVSP、右心室前壁厚度（RVAW）、右心室肥厚指数（RVHI）、右心室胶原容积分数（CVF）、远端肺动脉壁厚度比（WT）、肺动脉壁面积比（WA）增加，肺泡灌洗液白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α增加，右心室内径（RVID）降低（P<0.05）；与HPH组和HPH+agomir control组相比，HPH+miR-381-3p agomir组RVSP、RVAW、RVHI、右心室CVF、WT、WA，肺泡灌洗液IL-1β、IL-6、TNF-α降低，RVID增加（P<0.05）。数据库预测了miR-381-3p的下游靶基因，MuRF1是潜在的作用靶点，肌细胞中的细胞骨架通路（Cytoskeleton in muscle cells）居靶基因显著富集的首位。与WT-MuRF1+mimic control组相比，WT-MuRF1+miR-381-3p mimic组萤光素酶活性降低（P<0.05）；而Mut-MuRF1+mimic control组和Mut-MuRF1+miR-381-3p mimic组间差异无统计学意义。结论过表达miR-381-3p可改善HPH小鼠心肺损伤，其机制可能与miR-381-3p靶向抑制MuRF1有关。

关键词: 低氧, 高血压, 肺性, 肌肉环指蛋白1, 微小RNA-381-3p, 心肌纤维化, 肺血管重塑

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

中图分类号:

R544.16，R541.3

图/表 9

表1 引物序列

Tab.1 Primer sequences

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

表2 各组RVSP水平和右心功能指标比较（n=15，$\bar{x} \pm s$）

Tab.2 Comparison of RVSP level and right heart function index between the four groups

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^＊＊

图1 天狼星染色检测各组小鼠右心室心肌胶原沉积情况（×200）

Fig.1 The collagen deposition of right ventricle detected by Sirius red staining in the four groups (×200)

图2 各组小鼠远端肺动脉病理变化（HE染色，×400）黄色箭头指示远端肺小动脉。

Fig.2 Pathological changes of distal pulmonary artery in each group (HE staining, ×400)

表3 各组小鼠远端肺动脉WT和WA的比较（n=15，%，$\bar{x} \pm s$）

Tab.3 Comparison of WT and WA in distal pulmonary artery of mice between the four groups

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^＊＊

表4 各组小鼠肺泡灌洗液炎性因子水平比较（n=15，ng/L，$\bar{x} \pm s$）

Tab.4 Comparison of inflammatory factors in bronchoalveolar lavage fluid of mice hetween the four groups

组别	IL-1β	IL-6	TNF-α
NC组	287.65±26.46	18.63±9.84	94.82±46.36
HPH组	1 201.47±198.39^a	180.27±20.39^a	782.38±179.04^a
HPH+agomir control组	1 061.73±153.12^a	171.48±26.31^a	826.19±264.41^a
HPH+miR-381-3p agomir组	514.46±61.41^bc	67.72±8.91^bc	373.59±101.06^bc
F	169.200^＊＊	293.600^＊＊	62.580^＊＊

图3 miR-381-3p靶点分析及KEGG分析流程图

Fig.3 Flow chart of miR-381-3p target analysis and KEGG analysis

图4 DAVID在线KEGG通路富集分析

Fig.4 DAVID online results of KEGG enrichment pathway analysis

图5 miR-381-3p与MuRF1 3'-UTR的结合位点

Fig.5 Binding site of miR-381-3p to MuRF1 3'-UTR

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miRNA-381-3p/MuRF1轴对低氧性肺动脉高压小鼠心肺损伤的影响

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

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