沉默DDX17通过降低mTORC1活性抑制肺动脉平滑肌细胞的增殖和迁移

doi:10.12122/j.issn.1673-4254.2025.11.20

摘要/Abstract

摘要：

目的探讨DDX17在肺动脉高压发生发展中对肺动脉平滑肌细胞（PASMCs）增殖和迁移的作用及机制。方法 PASMCs体外培养，分别经低氧模拟肺动脉高压体外环境、转染si-Ddx17干扰DDX17表达、胰岛素恢复mTORC1活性。设置分组：PASMCs正常培养组（CON组）；PASMCs缺氧组（Hy组）；正常培养组PASMCs+si-Ddx17（si-Ddx17组）；缺氧组PASMCs+si-Ddx17（Hy+si-Ddx17组）；缺氧组PASMCs+胰岛素（Hy+insulin组）；缺氧组PASMCs+si-Ddx17+胰岛素（Hy+si-Ddx17+insulin组）。增殖细胞核抗原（Ki-67）免疫荧光染色检测PASMCs增殖能力，划痕及transwell实验检测PASMCs迁移能力。Western blotting检测DDX17、4EBP1、S6、p-4EBP1、p-S6蛋白水平变化。通过腹腔注射野百合碱（MCT）诱导小鼠肺动脉高压形成、转染AD-Ddx17i沉默体内DDX17的表达后，采用HE染色实验评估小鼠肺血管的形态。结果与CON组相比，Hy组PASMCs的增殖及迁移能力增强（P<0.01）且p-4EBP1、p-S6蛋白的表达升高（P<0.01）；与Hy组相比，Hy+si-Ddx17组PASMCs的增殖及迁移能力明显降低（P<0.05）；与Hy组相比，Hy+si-Ddx17组p-4EBP1、p-S6蛋白的表达降低（P<0.05）。与Hy+si-Ddx17组相比，Hy+si-Ddx17+insulin组增殖及迁移能力增强（P<0.05）。在MCT诱导的肺动脉高压小鼠体内转染AD-Ddx17i后，肺血管管腔狭窄及内膜增生均有所减轻。结论本研究发现在缺氧诱导的PASMCs和MCT诱导的肺动脉高压小鼠中DDX17的表达均升高，干扰DDX17的表达后可明显抑制PASMCs的增殖、迁移及肺动脉高压小鼠的肺血管的重构，其机制与mTORC1活性降低有关。

关键词: DDX17, 肺动脉高压, 肺动脉平滑肌细胞, 增殖, 迁移, mTORC1

Abstract:

Objective To investigate the mechanism of DDX17 for regulating proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) during the development of pulmonary hypertension (PH). Methods In murine PASMCs cultured under normoxic or hypoxic conditions, the effects of transfection with si-Ddx17 and insulin treatment, alone or in combination, on cell proliferation and migration were evaluated using Ki-67 immunofluorescence staining, scratch assay and Transwell assay. Western Blotting was performed to detect the changes in protein expression levels of DDX17, 4EBP1, S6, p-4EBP1, and p-S6. In a mouse model of PH induced by intraperitoneal injection of monocrotaline (MCT), the changes in pulmonary vasculature were examined using HE staining following tail vein injection of AD-Ddx17i. Results The PASMCs in hypoxic culture exhibited significantly enhanced cell proliferation and migration and protein expressions of p-4EBP1 and p-S6, and these changes were obviously reversed by transfection with si-Ddx17. Treatment with insulin significantly attenuated the effect of si-Ddx17 against hypoxic exposure-induced changes in PASMCs. In the mouse model of MCT-induced PH, transfection with AD-Ddx17i obviously alleviated pulmonary vascular stenosis and intimal hyperplasia. Conclusion The expression of DDX17 is elevated in hypoxia-induced PASMCs and PH mice, and silencing DDX17 significantly inhibits PASMC proliferation and migration in vitro and pulmonary vascular remodeling in PH mice by reducing mTORC1 activity.

Key words: DDX17, pulmonary hypertension, pulmonary artery smooth muscle cells, proliferation, migration, mTORC1

邓湘湘, 王嘉, 熊迷, 王挺, 杨永健, 李德, 孙雄山. 沉默DDX17通过降低mTORC1活性抑制肺动脉平滑肌细胞的增殖和迁移[J]. 南方医科大学学报, 2025, 45(11): 2475-2482.

Xiangxiang DENG, Jia WANG, Mi XIONG, Ting WANG, Yongjian YANG, De LI, Xiongshan SUN. Silencing DDX17 inhibits proliferation and migration of pulmonary arterial smooth muscle cells in vitro by decreasing mTORC1 activity[J]. Journal of Southern Medical University, 2025, 45(11): 2475-2482.

图/表 5

图1 低氧对 PASMCs 中 DDX17表达的影响

Fig.1 Effect of hypoxia on DDX17 mRNA (A) and protein expression level (B, C) in PASMCs (n=3, Mean±SD). **P<0.01 vs CON group.

图2 低氧条件下DDX17对PASMCs 增殖及迁移的影响

Fig.2 Effect of DDX17 silecing on proliferation and migration of PASMCs exposed to hypoxia (n=3, Mean±SD). A: DDX17 mRNA level in PASMCs. B, C: Protein expression level of DDX17 in PASMCs. D, E: Scratch healing assay for assessing changes in migration of PASMCs after the treatments (scale bar=100 µm. F, G: Transwell assay for assessing changes in migration ability of PASMCs after the treatments (scale bar=100 µm). H, I: Results of immunofluorescence staining of Ki-67 for assessing proliferative ability of PASMCs (scale bar=200 μm). *P<0.05, **P<0.01 vs CON group; #P<0.05, ##P<0.01 vs Hy group.

图3 DDX17对缺氧诱导的PASMCs中mTORC1活性的影响

Fig.3 Effect of DDX17 silencing on mTORC1 activity in hypoxia-induced PASMCs (n=3, Mean±SD). **P<0.01, ***P<0.001 vs CON group; #P<0.05 vs Hy group.

图4 mTORC1在DDX17沉默介导的PASMCs增殖和迁移抑制中的作用

Fig.4 Role of mTORC1 in DDX17 silencing-mediated inhibition of proliferation and migration of PASMCs (n=3, Mean±SD). A-C: Protein levels of p-4EBP1 and p-S6 in the cells detected using Western blotting. D, E: Scratch healing assay for assessing migration ability of PASMCs (scale bar=100 µm). F, G: Transwell assay for assessing migration ability of PASMCs (scale bar=100 µm). H, I: Immunofluorescence staining of Ki-67 for assessing proliferative ability of PASMCs (scale bar=200 µm). *P<0.05, **P<0.01 vs Hy group; #P <0.05, ##P<0.01 vs Hy+si-Ddx17 group.

图5 干扰DDX17表达减轻了MCT诱导的肺血管重构

Fig.5 DDX17 silencing ameliorates MCT-induced pulmonary vascular remodeling in mice (n=3, Mean±SD). A, B: Protein levels of DDX17 in the pulmonary artery of the mice detected using Western blotting. C: Morphological changes in pulmonary arteries of the mice (HE staining, ×40). D: Percentage of pulmonary wall thickness/total thickness in the mice in different groups; *P<0.05, ***P<0. 001 vs CON group; #P<0.05, ##P<0.01 vs MCT group.

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