A microRNA‑17‑5p/homeobox B13 axis participates in the phenotypic modulation of vascular smooth muscle cells

Yu,Tianchi; Wang,Tao; Kuang,Shifang; Zhao,Guoping; Zhou,Kun; Zhang,Hui

doi:10.3892/mmr.2021.12370

A microRNA‑17‑5p/homeobox B13 axis participates in the phenotypic modulation of vascular smooth muscle cells

Authors:
- Tianchi Yu
- Tao Wang
- Shifang Kuang
- Guoping Zhao
- Kun Zhou
- Hui Zhang
View Affiliations

Affiliations: Department of Vascular Surgery, Jiangning Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 211100, P.R. China, Center of Endoscopy, Jiangning Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 211100, P.R. China, Department of General Surgery, Jiangning Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu 211100, P.R. China
Published online on: August 16, 2021 https://doi.org/10.3892/mmr.2021.12370
Article Number: 731
Copyright: © Yu 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

Vascular smooth muscle cells (VSMCs) serve a decisive role in intimal hyperplasia, a common pathophysiological process that leads to numerous vascular disorders. The present study aimed to investigate the unknown mechanisms underlying VSMC phenotypic modulation and identified a novel microRNA (miRNA/miR)‑17‑5p/homeobox B13 (HOXB13) axis involved in the phenotypic switching, proliferation and migration of VSMCs. VSMCs were isolated from the thoracic aorta of Sprague‑Dawley rats, cell proliferation was determined by Cell Counting Kit‑8 (CCK‑8) assay, cell migration was examined by Transwell migration assay and gene expression was detected using reverse transcription‑quantitative PCR and western blot analyses. It was firstly found that incubation with platelet‑derived growth factor‑BB (PDGF‑BB) recombinant protein resulted in a significant increase in HOXB13 expression in VSMCs. Using multiple miRNA prediction tools, miR‑17‑5p was identified as a potential regulator for HOXB13, since it had a 7‑base perfect binding site and a 5‑base imperfect binding site with the 3'‑untranslated region of HOXB13 mRNA, and these sequences were highly conserved across species. The regulatory effect of miR‑17‑5p on HOXB13 was validated using luciferase reporter assays. The expression level of miR‑17‑5p was increased in VSMCs under PDGF‑BB stimulation, and was negatively correlated with HOXB13 mRNA and protein expression. Moreover, the miR‑17‑5p mimics significantly inhibited the proliferation and migration of VSMCs, while antagomiR‑17‑5p showed the opposite effects, which could be abolished by HOXB13 knockdown. The miR‑17‑5p/HOXB13 axis also regulated the expression levels of the markers of differentiated VSMCs (α‑smooth muscle actin, transgelin and smoothelin), proliferating cell nuclear antigen and cell migration proteins, including MMP‑2 and ‑9. In conclusion, the present study demonstrated that miR‑17‑5p inhibited the phenotypic modulation VSMCs stimulated by PDGF‑BB by downregulating HOXB13, indicating that these factors may be potential therapeutic targets for intimal hyperplasia.

View Figures

View References

1	Wang G, Jacquet L, Karamariti E and Xu Q: Origin and differentiation of vascular smooth muscle cells. J Physiol. 593:3013–3030. 2015. View Article : Google Scholar : PubMed/NCBI
2	Alexander MR and Owens GK: Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease. Annu Rev Physiol. 74:13–40. 2012. View Article : Google Scholar : PubMed/NCBI
3	Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI
4	Ha M and Kim VN: Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 15:509–524. 2014. View Article : Google Scholar : PubMed/NCBI
5	Mellis D and Caporali A: MicroRNA regulation of vascular function. Vasc Biol. 1:H41–H46. 2019. View Article : Google Scholar : PubMed/NCBI
6	Wang D and Atanasov AG: The microRNAs regulating vascular smooth muscle cell proliferation: A minireview. Int J Mol Sci. 20:3242019. View Article : Google Scholar : PubMed/NCBI
7	Holland PW: Evolution of homeobox genes. Wiley Interdiscip Rev Dev Biol. 2:31–45. 2013. View Article : Google Scholar : PubMed/NCBI
8	Aspuria PJ, Cheon DJ, Gozo MC, Beach JA, Recouvreux MS, Walts AE, Karlan BY and Orsulic S: HOXB13 controls cell state through super-enhancers. Exp Cell Res. 393:1120392020. View Article : Google Scholar : PubMed/NCBI
9	Cantile M, Schiavo G, Terracciano L and Cillo C: Homeobox genes in normal and abnormal vasculogenesis. Nutr Metab Cardiovasc Dis. 18:651–658. 2008. View Article : Google Scholar : PubMed/NCBI
10	Attard G, Parker C, Eeles RA, Schröder F, Tomlins SA, Tannock I, Drake CG and de Bono JS: Prostate cancer. Lancet. 387:70–82. 2016. View Article : Google Scholar : PubMed/NCBI
11	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
12	Li W, Deng P, Wang J, Li Z and Zhang H: MiR-17 knockdown promotes vascular smooth muscle cell phenotypic modulation through upregulated interferon regulator Factor 9 expression. Am J Hypertens. 33:1119–1126. 2020. View Article : Google Scholar : PubMed/NCBI
13	Lee MH, Kwon BJ, Koo MA, You KE and Park JC: Mitogenesis of vascular smooth muscle cell stimulated by platelet-derived growth factor-bb is inhibited by blocking of intracellular signaling by epigallocatechin-3-O-gallate. Oxid Med Cell Longev. 2013:8279052013. View Article : Google Scholar : PubMed/NCBI
14	Risinger GM Jr, Hunt TS, Updike DL, Bullen EC and Howard EW: Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors. J Biol Chem. 281:25915–25925. 2006. View Article : Google Scholar : PubMed/NCBI
15	Hsuan CF, Lu YC, Tsai IT, Houng JY, Wang SW, Chang TH, Chen YL and Chang CC: Glossogyne tenuifolia attenuates proliferation and migration of vascular smooth muscle cells. Molecules. 25:58322020. View Article : Google Scholar : PubMed/NCBI
16	Xin H, Wang Z, Wu S, Wang P, Tao X, Xu C and You L: Calcified decellularized arterial scaffolds impact vascular smooth muscle cell transformation via downregulating α-SMA expression and upregulating OPN expression. Exp Ther Med. 18:705–710. 2019.PubMed/NCBI
17	Je HD and Sohn UD: SM22alpha is required for agonist-induced regulation of contractility: Evidence from SM22alpha knockout mice. Mol Cells. 23:175–181. 2007.PubMed/NCBI
18	van Eys GJ, Niessen PM and Rensen SS: Smoothelin in vascular smooth muscle cells. Trends Cardiovasc Med. 17:26–30. 2007. View Article : Google Scholar : PubMed/NCBI
19	Newby AC: Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates. Cardiovasc Res. 69:614–624. 2006. View Article : Google Scholar : PubMed/NCBI
20	Dzau VJ, Braun-Dullaeus RC and Sedding DG: Vascular proliferation and atherosclerosis: New perspectives and therapeutic strategies. Nat Med. 8:1249–1256. 2002. View Article : Google Scholar : PubMed/NCBI
21	Xu F, Ahmed AS, Kang X, Hu G, Liu F, Zhang W and Zhou J: MicroRNA-15b/16 attenuates vascular neointima formation by promoting the contractile phenotype of vascular smooth muscle through targeting YAP. Arterioscler Thromb Vasc Biol. 35:2145–2152. 2015. View Article : Google Scholar : PubMed/NCBI
22	Yang F, Chen Q, He S, Yang M, Maguire EM, An W, Afzal TA, Luong LA, Zhang L and Xiao Q: miR-22 Is a novel mediator of vascular smooth muscle cell phenotypic modulation and neointima formation. Circulation. 137:1824–1841. 2018. View Article : Google Scholar : PubMed/NCBI
23	Montezano AC, Lopes RA, Neves KB, Rios F and Touyz RM: Isolation and culture of vascular smooth muscle cells from small and large vessels. Methods Mol Biol. 1527:349–354. 2017. View Article : Google Scholar : PubMed/NCBI
24	Li P, Zhu N, Yi B, Wang N, Chen M, You X, Zhao X, Solomides CC, Qin Y and Sun J: MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation. Circ Res. 113:1117–1127. 2013. View Article : Google Scholar : PubMed/NCBI
25	Ohnaka M, Marui A, Yamahara K, Minakata K, Yamazaki K, Kumagai M, Masumoto H, Tanaka S, Ikeda T and Sakata R: Effect of microRNA-145 to prevent vein graft disease in rabbits by regulation of smooth muscle cell phenotype. J Thorac Cardiovasc Surg. 148:676–682.e2. 2014. View Article : Google Scholar : PubMed/NCBI
26	Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, Muth AN, Lee TH, Miano JM, Ivey KN and Srivastava D: miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature. 460:705–710. 2009. View Article : Google Scholar : PubMed/NCBI
27	Vengrenyuk Y, Nishi H, Long X, Ouimet M, Savji N, Martinez FO, Cassella CP, Moore KJ, Ramsey SA, Miano JM and Fisher EA: Cholesterol loading reprograms the microRNA-143/145-myocardin axis to convert aortic smooth muscle cells to a dysfunctional macrophage-like phenotype. Arterioscler Thromb Vasc Biol. 35:535–546. 2015. View Article : Google Scholar : PubMed/NCBI
28	Yang D, Sun C, Zhang J, Lin S, Zhao L, Wang L, Lin R, Lv J and Xin S: Proliferation of vascular smooth muscle cells under inflammation is regulated by NF-κB p65/microRNA-17/RB pathway activation. Int J Mol Med. 41:43–50. 2018.PubMed/NCBI
29	Leeper NJ, Raiesdana A, Kojima Y, Chun HJ, Azuma J, Maegdefessel L, Kundu RK, Quertermous T, Tsao PS and Spin JM: MicroRNA-26a is a novel regulator of vascular smooth muscle cell function. J Cell Physiol. 226:1035–1043. 2011. View Article : Google Scholar : PubMed/NCBI
30	Tan J, Yang L, Liu C and Yan Z: MicroRNA-26a targets MAPK6 to inhibit smooth muscle cell proliferation and vein graft neointimal hyperplasia. Sci Rep. 7:466022017. View Article : Google Scholar : PubMed/NCBI
31	Chen Z, Wu J, Yang C, Fan P, Balazs L, Jiao Y, Lu M, Gu W, Li C, Pfeffer LM, et al: DiGeorge syndrome critical region 8 (DGCR8) protein-mediated microRNA biogenesis is essential for vascular smooth muscle cell development in mice. J Biol Chem. 287:19018–19028. 2012. View Article : Google Scholar : PubMed/NCBI
32	Chen T, Zhou Q, Tang H, Bozkanat M, Yuan JX, Raj JU and Zhou G: miR-17/20 Controls prolyl hydroxylase 2 (PHD2)/Hypoxia-Inducible Factor 1 (HIF1) to regulate pulmonary artery smooth muscle cell proliferation. J Am Heart Assoc. 5:e0045102016. View Article : Google Scholar : PubMed/NCBI
33	Wang X, Sun Y, Xu T, Qian K, Huang B, Zhang K, Song Z, Qian T, Shi J and Li L: HOXB13 promotes proliferation, migration, and invasion of glioblastoma through transcriptional upregulation of lncRNA HOXC-AS3. J Cell Biochem. 120:15527–15537. 2019. View Article : Google Scholar : PubMed/NCBI
34	Ewing CM, Ray AM, Lange EM, Zuhlke KA, Robbins CM, Tembe WD, Wiley KE, Isaacs SD, Johng D, Wang Y, et al: Germline mutations in HOXB13 and prostate-cancer risk. N Engl J Med. 366:141–149. 2012. View Article : Google Scholar : PubMed/NCBI
35	Nguyen NUN, Canseco DC, Xiao F, Nakada Y, Li S, Lam NT, Muralidhar SA, Savla JJ, Hill JA, Le V, et al: A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes. Nature. 582:271–276. 2020. View Article : Google Scholar : PubMed/NCBI
36	Zuo L, Tan T, Wei C, Wang H, Tan L, Hao Y, Qian J, Chen Y and Wu C: HOXB13 expression is correlated with hepatic inflammatory activity of patients with hepatic fibrosis. J Mol Histol. 51:183–189. 2020. View Article : Google Scholar : PubMed/NCBI