Expression of the microRNA‑30 family in pulmonary arterial hypertension and the role of microRNA‑30d‑5p in the regulation of pulmonary arterial smooth muscle cell toxicity and apoptosis

Hu,Fan; Liu,Hanmin; Wang,Chuan; Li,Hanwen; Qiao,Lina

doi:10.3892/etm.2021.11031

Expression of the microRNA‑30 family in pulmonary arterial hypertension and the role of microRNA‑30d‑5p in the regulation of pulmonary arterial smooth muscle cell toxicity and apoptosis

Authors:
- Fan Hu
- Hanmin Liu
- Chuan Wang
- Hanwen Li
- Lina Qiao
View Affiliations

Affiliations: Department of Pediatrics, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, P.R. China, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610041, P.R. China
Published online on: December 2, 2021 https://doi.org/10.3892/etm.2021.11031
Article Number: 108
Copyright: © Hu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The biological processes of pulmonary artery vascular smooth muscle cells (PA‑SMCs) and pulmonary artery endothelial cells in pulmonary arterial hypertension (PAH) are generally abnormal, with increased levels of proliferation and reduced levels of apoptosis. Although microRNAs (miRNAs/miRs) participate in a number of biological processes in a variety of diseases, such as tumors and infections, studies on the association between miRNAs and PAH are limited. In the present study, blood samples were collected from 6 patients with patent ductus arteriosus. The experimental group included 3 patients with severe PAH, while the control group included 3 patients without PAH. Microarray technology was used to detect the presence of any associated miRNAs. Moreover, a rat PAH model was established via left lung resection followed by monocrotaline injection, involving a total of 8 rats in the PAH group and 8 untreated rat in the control group. Reverse transcription‑quantitative PCR was performed to verify the expression levels of the miR‑30 family in the animal model. miR‑30d‑5p mimics and anti‑miR‑30d‑5p were transfected into primary cultured PA‑SMCs. Levels of cytotoxicity and cell apoptosis were examined, and Notch‑3 expression levels were studied using western blotting. The results of the present study demonstrated that miR‑30d‑5p expression was downregulated in both patient blood and animal models of the PAH group compared with control groups. In primary cultured PA‑SMCs, overexpression of miR‑30d‑5p attenuated the platelet‑derived growth factor‑induced toxicity of PA‑SMCs, while knockdown of miR‑30d‑5p resulted in the increased toxicity of PA‑SMCs compared with control group. The apoptosis rate of PA‑SMCs increased with the overexpression of miR‑30d‑5p compared with control group. Moreover, the expression levels of Notch‑3 in the miR‑30d‑5p group were significantly reduced compared with the anti‑miR‑30d‑5p and miR‑NC groups. In total, 10 circulating miRNAs that may be associated with PAH were discovered in the present study. Moreover, the expression of the miR‑30 family was verified in animal models in vivo, and seven miRNAs in this family were discovered that may be associated with PAH. Additionally, miR‑30d‑5p was downregulated in both patients with PAH and animal models compared with control groups. Thus, the results of the present study demonstrated that the regulatory mechanism underlying PA‑SMCs may be via the Notch‑3 signaling pathway.

View Figures

View References

1	Hu F, Liu C, Liu H, Xie L and Yu L: Ataxia-telangiectasia mutated (ATM) protein signaling participates in development of pulmonary arterial hypertension in rats. Med Sci Monit. 23:4391–4400. 2017.PubMed/NCBI View Article : Google Scholar
2	Goldberg AB, Mazur W and Kalra DK: Pulmonary hypertension: Diagnosis, imaging techniques, and novel therapies. Cardiovasc Diagn Ther. 7:405–417. 2017.PubMed/NCBI View Article : Google Scholar
3	Hansmann G: Pulmonary hypertension in infants, children, and young adults. J Am Coll Cardiol. 69:2551–2569. 2017.PubMed/NCBI View Article : Google Scholar
4	McClenaghan C, Woo KV and Nichols CG: Pulmonary hypertension and ATP-sensitive potassium channels. Hypertension. 74:14–22. 2019.PubMed/NCBI View Article : Google Scholar
5	Elia D, Caminati A, Zompatori M, Cassandro R, Lonati C, Luisi F, Pelosi G, Provencher S and Harari S: Pulmonary hypertension and chronic lung disease: Where are we headed? Eur Respir Rev. 28(31636088)2019.PubMed/NCBI View Article : Google Scholar
6	Singh I, Oliveira RKF, Naeije R, Rahaghi FN, Oldham WM, Systrom DM and Waxman AB: Pulmonary vascular distensibility and early pulmonary vascular remodeling in pulmonary hypertension. Chest. 156:724–732. 2019.PubMed/NCBI View Article : Google Scholar
7	Ye J, Xu M, Tian X, Cai S and Zeng S: Research advances in the detection of miRNA. J Pharm Anal. 9:217–226. 2019.PubMed/NCBI View Article : Google Scholar
8	Lin S and Gregory RI: MicroRNA biogenesis pathways in cancer. Nat Rev Cancer. 15:321–333. 2015.PubMed/NCBI View Article : Google Scholar
9	Wang S, Cao W, Gao S, Nie X, Zheng X, Xing Y, Chen Y, Bao H and Zhu D: TUG1 Regulates Pulmonary Arterial Smooth Muscle Cell Proliferation in Pulmonary Arterial Hypertension. Can J Cardiol. 35:1534–1545. 2019.PubMed/NCBI View Article : Google Scholar
10	Wang Y, Pandey RN, York AJ, Mallela J, Nichols WC, Hu YC, Molkentin JD, Wikenheiser-Brokamp KA and Hegde RS: The EYA3 tyrosine phosphatase activity promotes pulmonary vascular remodeling in pulmonary arterial hypertension. Nat Commun. 10(4143)2019.PubMed/NCBI View Article : Google Scholar
11	Ruffenach G, Umar S, Vaillancourt M, Hong J, Cao N, Sarji S, Moazeni S, Cunningham CM, Ardehali A, Reddy ST, et al: Histological hallmarks and role of Slug/PIP axis in pulmonary hypertension secondary to pulmonary fibrosis. EMBO Mol Med. 11(e10061)2019.PubMed/NCBI View Article : Google Scholar
12	Zheng Y, Lu X, Xu L, Chen Z, Li Q and Yuan J: MicroRNA-675 promotes glioma cell proliferation and motility by negatively regulating retinoblastoma 1. Hum Pathol. 69:63–71. 2017.PubMed/NCBI View Article : Google Scholar
13	Cheleschi S, Tenti S, Mondanelli N, Corallo C, Barbarino M, Giannotti S, Gallo I, Giordano A and Fioravanti A: MicroRNA-34a and MicroRNA-181a mediate visfatin-induced apoptosis and oxidative stress via NF-κB pathway in human osteoarthritic chondrocytes. Cells. 8(874)2019.PubMed/NCBI View Article : Google Scholar
14	Wang H, Tan Z, Hu H, Liu H, Wu T, Zheng C, Wang X, Luo Z, Wang J, Liu S, et al: MicroRNA-21 promotes breast cancer proliferation and metastasis by targeting LZTFL1. BMC Cancer. 19(738)2019.PubMed/NCBI View Article : Google Scholar
15	Liu T, Zou XZ, Huang N, Ge XY, Yao MZ, Liu H, Zhang Z and Hu CP: Down-regulation of miR-204 attenuates endothelial-mesenchymal transition by enhancing autophagy in hypoxia-induced pulmonary hypertension. Eur J Pharmacol. 863(172673)2019.PubMed/NCBI View Article : Google Scholar
16	Babicheva A, Ayon RJ, Zhao T, Ek Vitorin JF, Pohl NM, Yamamura A, Yamamura H, Quinton BA, Ba M, Wu L, et al: MicroRNA-mediated downregulation of K⁺ channels in pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol. 318:L10–L26. 2020.PubMed/NCBI View Article : Google Scholar
17	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Δ Δ C(T)) Method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
18	Yin Y, Wu X, Yang Z, Zhao J, Wang X, Zhang Q, Yuan M, Xie L, Liu H and He Q: The potential efficacy of R8-modified paclitaxel-loaded liposomes on pulmonary arterial hypertension. Pharm Res. 30:2050–2062. 2013.PubMed/NCBI View Article : Google Scholar
19	Liu YF, Spinelli A, Sun LY, Jiang M, Singer DV, Ginnan R, Saddouk FZ, Van Riper D and Singer HA: MicroRNA-30 inhibits neointimal hyperplasia by targeting Ca(2+)/calmodulin-dependent protein kinase IIδ (CaMKIIδ). Sci Rep. 6(26166)2016.PubMed/NCBI View Article : Google Scholar
20	Morris HE, Neves KB, Montezano AC, MacLean MR and Touyz RM: Notch3 signalling and vascular remodelling in pulmonary arterial hypertension. Clin Sci (Lond). 133:2481–2498. 2019.PubMed/NCBI View Article : Google Scholar
21	Li L, Zhu X, Shou T, Yang L, Cheng X, Wang J, Deng L and Zheng Y: MicroRNA-28 promotes cell proliferation and invasion in gastric cancer via the PTEN/PI3K/AKT signalling pathway. Mol Med Rep. 17:4003–4010. 2018.PubMed/NCBI View Article : Google Scholar
22	Yang Z, Xu J, Zhu R and Liu L: Down-regulation of miRNA-128 contributes to neuropathic pain following spinal cord injury via activation of P38. Med Sci Monit. 23:405–411. 2017.PubMed/NCBI View Article : Google Scholar
23	Mundalil Vasu M, Anitha A, Thanseem I, Suzuki K, Yamada K, Takahashi T, Wakuda T, Iwata K, Tsujii M, Sugiyama T and Mori N: Serum microRNA profiles in children with autism. Mol Autism. 5(40)2014.PubMed/NCBI View Article : Google Scholar
24	Zhang K, Wu X, Wang J, Lopez J, Zhou W, Yang L, Wang SE, Raz DJ and Kim JY: Circulating miRNA profile in esophageal adenocarcinoma. Am J Cancer Res. 6:2713–2721. 2016.PubMed/NCBI
25	Yang M, Zhai X, Ge T, Yang C and Lou G: miR-181a-5p Promotes Proliferation and Invasion and Inhibits Apoptosis of Cervical Cancer Cells via Regulating Inositol Polyphosphate-5-Phosphatase A (INPP5A). Oncol Res. 26:703–712. 2018.PubMed/NCBI View Article : Google Scholar
26	Liu Z, Sun F, Hong Y, Liu Y, Fen M, Yin K, Ge X, Wang F, Chen X and Guan W: MEG2 is regulated by miR-181a-5p and functions as a tumour suppressor gene to suppress the proliferation and migration of gastric cancer cells. Mol Cancer. 16(133)2017.PubMed/NCBI View Article : Google Scholar
27	Li Q, Li Z, Wei S, Wang W, Chen Z, Zhang L, Chen L, Li B, Sun G, Xu J, et al: Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer. J Exp Clin Cancer Res. 36(59)2017.PubMed/NCBI View Article : Google Scholar
28	Liu J and Li SM: MiR-484 suppressed proliferation, migration, invasion and induced apoptosis of gastric cancer via targeting CCL-18. Int J Exp Pathol. 101:203–214. 2020.PubMed/NCBI View Article : Google Scholar
29	Wu J, He Y, Luo Y, Zhang L, Lin H, Liu X, Liu B, Liang C, Zhou Y and Zhou J: miR-145-5p inhibits proliferation and inflammatory responses of RMC through regulating AKT/GSK pathway by targeting CXCL16. J Cell Physiol. 233:3648–3659. 2018.PubMed/NCBI View Article : Google Scholar
30	Ma Q, Wang Y, Zhang H and Wang F: miR-1290 contributes to colorectal cancer cell proliferation by targeting INPP4B. Oncol Res. 26:1167–1174. 2018.PubMed/NCBI View Article : Google Scholar
31	Armstrong DA, Nymon AB, Ringelberg CS, Lesseur C, Hazlett HF, Howard L, Marsit CJ and Ashare A: Pulmonary microRNA profiling: implications in upper lobe predominant lung disease. Clin Epigenetics. 9(56)2017.PubMed/NCBI View Article : Google Scholar
32	Nakamura A, Rampersaud YR, Sharma A, Lewis SJ, Wu B, Datta P, Sundararajan K, Endisha H, Rossomacha E, Rockel JS, et al: Identification of microRNA-181a-5p and microRNA-4454 as mediators of facet cartilage degeneration. JCI Insight. 1(e86820)2016.PubMed/NCBI View Article : Google Scholar
33	Chen F, Yang J, Li Y and Wang H: Circulating microRNAs as novel biomarkers for heart failure. Hellenic J Cardiol. 59:209–214. 2018.PubMed/NCBI View Article : Google Scholar
34	Wang A, Kwee LC, Grass E, Neely ML, Gregory SG, Fox KAA, Armstrong PW, White HD, Ohman EM, Roe MT, et al: Whole blood sequencing reveals circulating microRNA associations with high-risk traits in non-ST-segment elevation acute coronary syndrome. Atherosclerosis. 261:19–25. 2017.PubMed/NCBI View Article : Google Scholar
35	Liang L, Zeng JH, Wang JY, He RQ, Ma J, Chen G, Cai XY and Hu XH: Down-regulation of miR-26a-5p in hepatocellular carcinoma: A qRT-PCR and bioinformatics study. Pathol Res Pract. 213:1494–1509. 2017.PubMed/NCBI View Article : Google Scholar
36	Zhao F, Qu Y, Zhu J, Zhang L, Huang L, Liu H, Li S and Mu D: miR-30d-5p plays an important role in autophagy and apoptosis in developing rat brains after hypoxic-ischemic injury. J Neuropathol Exp Neurol. 76:709–719. 2017.PubMed/NCBI View Article : Google Scholar
37	Song Y, Song C and Yang S: Tumor-Suppressive Function of miR-30d-5p in Prostate Cancer Cell Proliferation and Migration by Targeting NT5E. Cancer Biother Radiopharm. 33:203–211. 2018.PubMed/NCBI View Article : Google Scholar
38	Jia K, Shi P, Han X, Chen T, Tang H and Wang J: Diagnostic value of miR-30d-5p and miR-125b-5p in acute myocardial infarction. Mol Med Rep. 14:184–194. 2016.PubMed/NCBI View Article : Google Scholar
39	Li WF, Dai H, Ou Q, Zuo GQ and Liu CA: Overexpression of microRNA-30a-5p inhibits liver cancer cell proliferation and induces apoptosis by targeting MTDH/PTEN/AKT pathway. Tumour Biol. 37:5885–5895. 2016.PubMed/NCBI View Article : Google Scholar
40	Wang C, Cai L, Liu J, Wang G, Li H, Wang X, Xu W, Ren M, Feng L, Liu P, et al: MicroRNA-30a-5p inhibits the growth of renal cell carcinoma by modulating GRP78 expression. Cell Physiol Biochem. 43:2405–2419. 2017.PubMed/NCBI View Article : Google Scholar
41	Liu W, Li H, Wang Y, Zhao X, Guo Y, Jin J and Chi R: miR-30b-5p functions as a tumor suppressor in cell proliferation, metastasis and epithelial-to-mesenchymal transition by targeting G-protein subunit α-13 in renal cell carcinoma. Gene. 626:275–281. 2017.PubMed/NCBI View Article : Google Scholar
42	Qi B, Wang Y, Chen ZJ, Li XN, Qi Y, Yang Y, Cui GH, Guo HZ, Li WH and Zhao S: Down-regulation of miR-30a-3p/5p promotes esophageal squamous cell carcinoma cell proliferation by activating the Wnt signaling pathway. World J Gastroenterol. 23:7965–7977. 2017.PubMed/NCBI View Article : Google Scholar
43	Ceolotto G, Giannella A, Albiero M, Kuppusamy M, Radu C, Simioni P, Garlaschelli K, Baragetti A, Catapano AL, Iori E, et al: miR-30c-5p regulates macrophage-mediated inflammation and pro-atherosclerosis pathways. Cardiovasc Res. 113:1627–1638. 2017.PubMed/NCBI View Article : Google Scholar
44	Cao JM, Li GZ, Han M, Xu HL and Huang KM: miR-30c-5p suppresses migration, invasion and epithelial to mesenchymal transition of gastric cancer via targeting MTA1. Biomed Pharmacother. 93:554–560. 2017.PubMed/NCBI View Article : Google Scholar
45	Xu G, Cai J, Wang L, Jiang L, Huang J, Hu R and Ding F: MicroRNA-30e-5p suppresses non-small cell lung cancer tumorigenesis by regulating USP22-mediated Sirt1/JAK/STAT3 signaling. Exp Cell Res. 362:268–278. 2018.PubMed/NCBI View Article : Google Scholar
46	Qin X, Li C, Guo T, Chen J, Wang HT, Wang YT, Xiao YS, Li J, Liu P, Liu ZS, et al: Upregulation of DARS2 by HBV promotes hepatocarcinogenesis through the miR-30e-5p/MAPK/NFAT5 pathway. J Exp Clin Cancer Res. 36(148)2017.PubMed/NCBI View Article : Google Scholar
47	Wang K, Chen M and Wu W: Analysis of microRNA (miRNA) expression profiles reveals 11 key biomarkers associated with non-small cell lung cancer. World J Surg Oncol. 15(175)2017.PubMed/NCBI View Article : Google Scholar
48	Coskun E, Ercin M and Gezginci-Oktayoglu S: The role of epigenetic regulation and pluripotency-related microRNAs in differentiation of pancreatic stem cells to beta cells. J Cell Biochem. 119:455–467. 2018.PubMed/NCBI View Article : Google Scholar
49	Shukla K, Sharma AK, Ward A, Will R, Hielscher T, Balwierz A, Breunig C, Münstermann E, König R, Keklikoglou I, et al: MicroRNA-30c-2-3p negatively regulates NF-κB signaling and cell cycle progression through downregulation of TRADD and CCNE1 in breast cancer. Mol Oncol. 9:1106–1119. 2015.PubMed/NCBI View Article : Google Scholar
50	Mathew LK, Lee SS, Skuli N, Rao S, Keith B, Nathanson KL, Lal P and Simon MC: Restricted expression of miR-30c-2-3p and miR-30a-3p in clear cell renal cell carcinomas enhances HIF2α activity. Cancer Discov. 4:53–60. 2014.PubMed/NCBI View Article : Google Scholar
51	Jacquin S, Rincheval V, Mignotte B, Richard S, Humbert M, Mercier O, Londoño-Vallejo A, Fadel E and Eddahibi S: Inactivation of p53 is sufficient to induce development of pulmonary hypertension in rats. PLoS One. 10(e0131940)2015.PubMed/NCBI View Article : Google Scholar
52	Gao L, He RQ, Wu HY, Zhang TT, Liang HW, Ye ZH, Li ZY, Xie TT, Shi Q, Ma J, et al: Expression Signature and Role of miR-30d-5p in Non-Small Cell Lung Cancer: A comprehensive study based on in silico analysis of public databases and in vitro experiments. Cell Physiol Biochem. 50:1964–1987. 2018.PubMed/NCBI View Article : Google Scholar
53	Deng L, Blanco FJ, Stevens H, Lu R, Caudrillier A, McBride M, McClure JD, Grant J, Thomas M, Frid M, et al: MicroRNA-143 activation regulates smooth muscle and endothelial cell crosstalk in pulmonary arterial hypertension. Circ Res. 117:870–883. 2015.PubMed/NCBI View Article : Google Scholar
54	Caruso P, Dunmore BJ, Schlosser K, Schoors S, Dos Santos C, Perez-Iratxeta C, Lavoie JR, Zhang H, Long L, Flockton AR, et al: Identification of MicroRNA-124 as a major regulator of enhanced endothelial cell glycolysis in pulmonary arterial hypertension via PTBP1 (polypyrimidine tract binding protein) and pyruvate kinase M2. Circulation. 136:2451–2467. 2017.PubMed/NCBI View Article : Google Scholar
55	Cai Z, Li J, Zhuang Q, Zhang X, Yuan A, Shen L, Kang K, Qu B, Tang Y, Pu J, et al: miR-125a-5p ameliorates monocrotaline-induced pulmonary arterial hypertension by targeting the TGF-β1 and IL-6/STAT3 signaling pathways. Exp Mol Med. 50:1–11. 2018.PubMed/NCBI View Article : Google Scholar
56	Thistlethwaite PA, Li X and Zhang X: Notch signaling in pulmonary hypertension. Adv Exp Med Biol. 661:279–298. 2010.PubMed/NCBI View Article : Google Scholar
57	Skurikhin EG, Krupin VA, Pershina OV, Pan ES, Ermolaeva LA, Pakhomova AV, Rybalkina OY, Ermakova NN, Khmelevskaya ES, Vaizova OE, et al: Endothelial progenitor cells and Notch-1 signaling as markers of alveolar endothelium regeneration in pulmonary emphysema. Bull Exp Biol Med. 166:201–206. 2018.PubMed/NCBI View Article : Google Scholar
58	Guo ML, Kook YH, Shannon CE and Buch S: Notch3/VEGF-A axis is involved in TAT-mediated proliferation of pulmonary artery smooth muscle cells: Implications for HIV-associated PAH. Cell Death Discov. 4(22)2018.PubMed/NCBI View Article : Google Scholar