肺动脉平滑肌细胞外泌体上调miR-106b-5p增强肺动脉内皮细胞Warburg效应促进动脉型肺动脉高压的分子机制

doi:10.3969/j.issn.1006-5725.2023.17.007

摘要/Abstract

摘要：

目的探讨肺动脉平滑肌细胞与内皮细胞的胞间通讯在促进动脉型肺动脉高压（PAH）疾病进展中的作用机制。方法采集并分离健康个体（HC）和PAH患者外周血浆中的外泌体，并对其内的miRNAs进行微阵列测定和差异表达分析。体外缺氧诱导处理原代人肺动脉平滑肌细胞（hPASMCs）和原代肺动脉内皮细胞（hPAECs），分为常氧组和缺氧诱导组。生物信息学分析miR-106b-5p和USP32的序列互作位点。腺病毒转染过表达/敲低hPASMCs内miR-106b-5p，过表达/敲低hPAECs内PKM2。双荧光素酶报告基因分析确证二者的负作用方式。Western blot法测定胞内USP32、PKM2、GLUT1、HK2、LDHA蛋白质的表达水平。结果微阵列测定和差异表达分析结果显示，与HC组相比，PAH组外周血浆中外泌体内miR-106b-5p的水平明显上调。在体外，与常氧组相比，miR-106b-5p的水平在缺氧诱导组hPASMCs上清液外泌体内明显上调，而在hPAECs细胞上清液外泌体中则无此变化。miR-106b-5p负调控USP32的表达。与miR-NC组/Inhibitor-NC组相比，在hPASMCs中miR-106b-5p mimic/miR-106b-5p inhibitor处理后，胞内PKM2、GLUT1、HK2和LDHA蛋白质表达明显增强/抑制。与shRNA-NT组/vector组相比，在hPAECs中PKM2 shRNA/PKM2-OE处理后，胞内PKM2、GLUT1、HK2和LDHA蛋白质表达明显抑制/增强（P < 0.05）。结论 hPASMCs通过外泌体上调miR-106b-5p增强hPAECs胞内Warburg效应，在动脉型肺动脉高压中具有潜在的促进疾病进展的作用。

关键词: 动脉型肺动脉高压, 肺动脉平滑肌细胞, 肺动脉内皮细胞, 外泌体, miR-106b-5p, Warburg效应

Abstract:

Objective To explore the mechanisms of inter-cellular communication between pulmonary artery smooth muscle cells and pulmonary artery endothelial cells in promoting the progression of arterial pulmonary hypertension （PAH）. Methods Exosomes in peripheral blood of healthy individuals （HC） and PAH patients were collected and isolated， and miRNAs in the exosomes were determined by microarray and differential expression analysis. Primary human pulmonary artery smooth muscle cells （hPASMCs） and primary human pulmonary artery endothelial cells （hPAECs） were treated with normoxia or hypoxia in vitro. Bioinformatics analysis was performed to detect sequence interaction sites between miR-106b-5p and USP32. Adenovirus was transinfectedinto the hPASMCs of over-expressed/knocked-down miR-106b-5p， or into the hPAECs of the over-expressed/knocked-down of PKM2. Dual luciferase reporter assay was used for confirming the sequence interaction of miR-106b-5p and USP32. The levels of USP32， PKM2， GLUT1， HK2 and LDHA were determined by Western blot. Results By microarray assay and differential expression analysis， the level of miR-106b-5p in the peripheral blood-derived exosomes was significantly up-regulated in the PAH group as compared with HC group. Compared with normoxia group cells， the level of miR-106b-5p was significantly up-regulated in the hypoxia-induced hPASMCs cells’ supernatant exomsomes， but not in the exosomes of hPAECs cells’ supernatant. MiR-106b-5p negatively regulated the expression of USP32. Compared with the miR-NC group/inhibitor-NC group，the expressions of PKM2， GLUT1， HK2 and LDHA were significantly enhanced/inhibited after miR-106b-5p mimic/miR-106b-5p inhibitor was treated with hPASMCs. Compared with shRNA-NT/vector group， PKM2 shRNA/PKM2-OE treatment in hPAECs significantly inhibited/enhanced intracellular expressions of PKM2， GLUT1， HK2， and LDHA （P<0.05）. Conclusion HPASMCs enhances intracellular Warburg effect of hPAECs through up-regulating miR-106b-5p in exosomes， which has a potential role in promoting disease progression in arterial pulmonary hypertension.

Key words: arterial pulmonary hypertension, pulmonary smooth muscle cells, pulmonary endothelial cells, exosomes, miR-106b-5p, Warburg effect

中图分类号:

R544.1

艾丽菲热·买买提,高静,于子翔,马依彤. 肺动脉平滑肌细胞外泌体上调miR-106b-5p增强肺动脉内皮细胞Warburg效应促进动脉型肺动脉高压的分子机制[J]. 实用医学杂志, 2023, 39(17): 2190-2195.

Ailifeire MAIMAITI,Jing GAO,Zixiang YU,Yitong. MA. Up⁃regulated miR⁃106b⁃5p in exosomes derived from pulmonary artery smooth muscle cells enhances Warburg effect of pulmonary artery endothelial cells and promotes arterial pulmonary hypertension[J]. The Journal of Practical Medicine, 2023, 39(17): 2190-2195.

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miRNAs	差异表达基因表达倍数取Log	校正后的P值
显著上调
hsa-miR-8075	2.512 346	0.018 230
hsa-miR-6732-5p	2.301 753	0.002 374
hsa-miR-106b-5p	1.832 581	0.008 293
hsa-miR-6722	1.597 862	0.007 395
Hsa-miR-486-5p	0.915 943	0.029 723
显著下调
hsa-miR-6089	-3.237 94	0.004 267
hsa-miR-3178	-3.097 45	0.006 755
hsa-miR-6089	-2.884 35	0.004 756
hsa-miR-6090	-2.675 94	0.005 672
hsa-miR-4466	-2.443 76	0.006 758

组别	hPASMCs	hPAECs
常氧组	1.0ECssi	1.0ECssi
缺氧组	7.62Cssio	1.07Cssio
t值	34.01	2.38
P值	0.001	0.072

组别	USP32 WT	USP32 MUT
miR-NC组	1.0 ± 0.17	1.0 ± 0.10
miR-106b-5p mimic组	0.33 ± 0.03	1.04 ± 0.11
t值	7.47	2.05
P值	0.001	0.102

组别	USP32	PKM2
miR-NC组	1.0 ± 0.08	1.0 ± 0.06
miR-106b-5p mimic组	0.25 ± 0.04	2.23 ± 0.15
t值	16.71	24.36
P值	0.001	0.001

组别	PKM2	GLUT1	HK2	LDHA
miR-NC组	1.00 ± 0.11	1.00 ± 0.07	1.00 ± 0.14	1.00 ± 0.10
miR-106b-5p mimic组	2.28 ± 0.21^***	2.40 ± 0.13^***	1.95 ± 0.09^***	2.09 ± 0.14^***
t值	15.52	10.42	10.86	18.43
P值	0.001	0.001	0.001	0.001
Inhibitor-NC组	1.12 ± 0.07	1.05 ± 0.08	0.94 ± 0.10	1.11 ± 0.06
miR-106b-5p inhibitor组	0.22 ± 0.04^###	0.45 ± 0.03^###	0.51 ± 0.04^###	0.18 ± 0.02^###
t值	15.23	16.69	22.25	33.36
P值	0.001	0.001	0.001	0.001