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

Abstract

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

CLC Number:

R544.1

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.

Figures/Tables 8

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References 19

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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

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

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组别	PKM2	GLUT1	HK2	LDHA
shRNA-NT组	1.00 ± 0.13	1.00 ± 0.10	1.00 ± 0.18	1.00 ± 0.15
PKM2 shRNA组	0.27 ± 0.03^***	0.51 ± 0.04^***	0.23 ± 0.01^***	0.44 ± 0.03^*
t值	13.79	5.66	9.85	3.756
P值	0.001	0.005	0.001	0.02
vector组	1.06 ± 0.15	0.97 ± 0.11	1.08 ± 0.09	1.11 ± 0.07
PKM2-OE组	2.78 ± 0.24^##	2.26 ± 0.17^##	1.88 ± 0.13^###	2.47 ± 0.16^###
t值	7.107	6.35	16.58	11.88
P值	0.002	0.003	0.001	0.001

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