Silencing DDX17 inhibits proliferation and migration of pulmonary arterial smooth muscle cells in vitro by decreasing mTORC1 activity

doi:10.12122/j.issn.1673-4254.2025.11.20

Abstract

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

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.

Figures/Tables 5

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.

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.

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.

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.

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.

References 37

[1]	Simonneau G, Montani D, Celermajer DS, et al. Haemodynamic definitions and updated clinical classification of pulmonary hypertension[J]. Eur Respir J, 2019, 53(1): 1801913. doi：10.1183/13993003.01913-2018
[2]	Hoeper MM, Badagliacca R, Berger RMF, et al. Developed by the task force for the diagnosis and treatment of pulmonary hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS).[J]. Eur Heart J, 2023, 44(15):1312.
[3]	Maron BA, Galiè N. Pulmonary Arterial Hypertension Diagnosis, Treatment, and Clinical Management in the Contemporary Era[J]. JAMA Cardiol, 2016, 1(9): 1056-1065. doi：10.1001/jamacardio.2016.4471
[4]	Hurdman J, Condliffe R, Elliot CA, et al. ASPIRE registry: assessing the Spectrum of Pulmonary hypertension Identified at a REferral centre[J]. Eur Respir J, 2012, 39(4): 945-55. doi：10.1183/09031936.00078411
[5]	Zheng R, Xu T, Wang X, et al. Stem cell therapy in pulmonary hypertension: current practice and future opportunities[J]. Eur Respir Rev, 2023, 32(169): 230112. doi：10.1183/16000617.0112-2023
[6]	Xu K, Sun S, Yan M, et al. DDX5 and DDX17-multifaceted proteins in the regulation of tumorigenesis and tumor progression[J]. Front Oncol, 2022, 12: 943032. doi：10.3389/fonc.2022.943032
[7]	Wang SB, Narendran S, Hirahara S, et al. DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retro-transposon RNAs[J]. Sci Immunol, 2021, 6(66): eabi4493. doi：10.1126/sciimmunol.abi4493
[8]	Caretti G, Schiltz RL, Dilworth FJ, et al. The RNA helicases p68/p72 and the noncoding RNA sra are coregulators of MyoD and skeletal muscle differentiation[J]. Dev Cell, 2006, 11(4): 547-60. doi：10.1016/j.devcel.2006.08.003
[9]	Liu H, Gao X, Zhang W, et al. DDX17-mediated upregulation of CXCL8 promotes hepatocellular carcinoma progression via co-activating β-catenin/NF-κB complex[J]. Int J Biol Sci, 2025, 21(3): 1342-60. doi：10.7150/ijbs.104165
[10]	Fuller-Pace FV. DEAD box RNA helicase functions in cancer[J]. RNA Biol, 2013, 10(1): 121-32. doi：10.4161/rna.23312
[11]	Liu X, Li L, Geng C, et al. DDX17 promotes the growth and metastasis of lung adenocarcinoma[J]. Cell Death Discov, 2022, 8(1): 425. doi：10.1038/s41420-022-01215-x
[12]	Zhao G, Wang Q, Zhang Y, et al. DDX17 induces epithelial-mesenchymal transition and metastasis through the miR-149-3p/CYBRD1 pathway in colorectal cancer[J]. Cell Death Dis, 2023, 14(1): 1. doi：10.1038/s41419-022-05508-y
[13]	Zhou HZ, Li F, Cheng ST, et al. DDX17-regulated alternative splicing that produced an oncogenic isoform of PXN-AS1 to promote HCC metastasis[J]. Hepatology, 2022, 75(4): 847-65. doi：10.1002/hep.32195
[14]	Wu XC, Yan WG, Ji ZG, et al. Long noncoding RNA SNHG20 promotes prostate cancer progression via upregulating DDX17[J]. Arch Med Sci, 2021, 17(6): 1752-65.
[15]	Yan M, Gao J, Lan M, et al. DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure[J]. Signal Transduct Target Ther, 2024, 9(1): 127. doi：10.1038/s41392-024-01831-2
[16]	Lin B, Wang F, Wang J, et al. The protective role of p72 in doxorubicin-induced cardiomyocytes injury in vitro [J]. Mol Med Rep, 2016, 14(4): 3376-80. doi：10.3892/mmr.2016.5600
[17]	Gao R, Wang L, Bei Y, et al. Long noncoding RNA cardiac physiological hypertrophy-associated regulator induces cardiac physiological hypertrophy and promotes functional recovery after myocardial ischemia-reperfusion injury[J]. Circulation, 2021, 144(4): 303-17. doi：10.1161/circulationaha.120.050446
[18]	Liu GB, Cheng YX, Li HM, et al. Ghrelin promotes cardiomyocyte differentiation of adipose tissue-derived mesenchymal stem cells by DDX17-mediated regulation of the SFRP4/Wnt/β-catenin axis[J]. Mol Med Rep, 2023, 28(3): 164. doi：10.3892/mmr.2023.13050
[19]	He C, Zhang G, Lu Y, et al. DDX17 modulates the expression and alternative splicing of genes involved in apoptosis and proliferation in lung adenocarcinoma cells[J]. PeerJ, 2022, 10: e13895. doi：10.7717/peerj.13895
[20]	Sun X, Nakajima E, Norbrun C, et al. Chitinase 3 like 1 contributes to the development of pulmonary vascular remodeling in pulmonary hypertension[J]. JCI Insight, 2022, 7(18): e159578. doi：10.1172/jci.insight.159578
[21]	Pullamsetti SS, Savai R, Seeger W, et al. Translational advances in the field of pulmonary hypertension. from cancer biology to new pulmonary arterial hypertension therapeutics. targeting cell growth and proliferation signaling hubs[J]. Am J Respir Crit Care Med, 2017, 195(4): 425-37. doi：10.1164/rccm.201606-1226pp
[22]	Thenappan T, Ormiston ML, Ryan JJ, et al. Pulmonary arterial hypertension: pathogenesis and clinical management[J]. BMJ, 2018, 360: j5492. doi：10.1136/bmj.j5492
[23]	Song Y, Jia H, Ma Q, et al. The causes of pulmonary hypertension and the benefits of aerobic exercise for pulmonary hypertension from an integrated perspective[J]. Front Physiol, 2024, 15: 1461519. doi：10.3389/fphys.2024.1461519
[24]	Yang Q, Jankowsky E. ATP- and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box protein DED1[J]. Biochemistry, 2005, 44(41): 13591-601. doi：10.1021/bi0508946
[25]	Xing Z, Ma WK, Tran EJ. The DDX5/Dbp2 subfamily of DEAD-box RNA helicases[J]. Wiley Interdiscip Rev RNA, 2019, 10(2): e1519. doi：10.1002/wrna.1519
[26]	Kay BK, Williamson MP, Sudol M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains[J]. FASEB J, 2000, 14(2): 231-41. doi：10.1096/fasebj.14.2.231
[27]	Mori M, Triboulet R, Mohseni M, et al. Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer[J]. Cell, 2014, 156(5): 893-906. doi：10.1016/j.cell.2013.12.043
[28]	Sun Q, Chen X, Ma J, et al. Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth[J]. Proc Natl Acad Sci USA, 2011, 108(10): 4129-34. doi：10.1073/pnas.1014769108
[29]	Yang Q, Guan KL. Expanding mTOR signaling[J]. Cell Res, 2007, 17(8): 666-81. doi：10.1038/cr.2007.64
[30]	Ayuk SM, Abrahamse H. mTOR signaling pathway in cancer targets photodynamic therapy in vitro [J]. Cells, 2019, 8(5): E431. doi：10.3390/cells8050431
[31]	Houssaini A, Abid S, Derumeaux G, et al. Selective tuberous sclerosis complex 1 gene deletion in smooth muscle activates mammalian target of rapamycin signaling and induces pulmonary hypertension[J]. Am J Respir Cell Mol Biol, 2016, 55(3): 352-67. doi：10.1165/rcmb.2015-0339oc
[32]	He Y, Zuo C, Jia D, et al. Loss of DP1 aggravates vascular remodeling in pulmonary arterial hypertension via mTORC1 signaling[J]. Am J Respir Crit Care Med, 2020, 201(10): 1263-76. doi：10.1164/rccm.201911-2137oc
[33]	Goncharova EA, Simon MA, Yuan JX. mTORC1 in pulmonary arterial hypertension. At the crossroads between vasoconstriction and vascular remodeling [J] ?Am J Respir Crit Care Med, 2020, 201(10): 1177-9. doi：10.1164/rccm.202001-0087ed
[34]	Tang HY, Wu K, Wang J, et al. Pathogenic role of mTORC1 and mTORC2 in pulmonary hypertension[J]. JACC Basic Transl Sci, 2018, 3(6): 744-62. doi：10.1016/j.jacbts.2018.08.009
[35]	Wang G, Chen L, Lei X, et al. Role of FLCN phosphorylation in insulin-mediated mTORC1 activation and tumorigenesis[J]. Adv Sci: Weinh, 2023, 10(17): e2206826. doi：10.1002/advs.202206826
[36]	Yang H, Jiang X, Li B, et al. Mechanisms of mTORC1 activation by RHEB and inhibition by PRAS40[J]. Nature, 2017, 552(7685): 368-73. doi：10.1038/nature25023
[37]	Lai M, Zou W, Han Z, et al. Tsc1 regulates tight junction independent of mTORC1[J]. Proc Natl Acad Sci USA, 2021, 118(30): e2020891118. doi：10.1073/pnas.2020891118