Korean J Physiol Pharmacol.  2022 May;26(3):183-193. 10.4196/kjpp.2022.26.3.183.

Differentially expressed mRNAs and their upstream miR-491-5p in patients with coronary atherosclerosis as well as the function of miR-491-5p in vascular smooth muscle cells

Affiliations
  • 1Department of Cardiovascular Surgery, The Affiliated Huaian No. 1 People’s Hospital of Nanjing Medical University, Huaian, Jiangsu 223300, China

Abstract

MicroRNAs (miRNAs) regulate gene expression and are biomarkers for coronary atherosclerosis (AS). A novel miRNA-mRNA regulation network of coronary AS still needs to be disclosed. The aim of this study was to analyze potential mRNAs in coronary AS patients and the role of their upstream miR-491-5p in vascular smooth muscle cells (VSMCs). We first confirmed top ten mRNAs according to the analysis from Gene Expression Omnibus database (GSE132651) and examined the expression levels of them in the plaques and serum from AS patients. Five mRNAs (UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2) presented significantly abnormal expression in both plaques and serum from AS patients, compared with that in the control groups. Subsequently, they were predicted to be targeted by 11 miRNAs by bioinformatics analysis. Among all the potential upstream miRNAs, only miR-491-5p was abnormally expressed in the plaques and serum from AS patients. Notably, miR-491-5p overexpression inhibited viability and migration, and significantly increased the expression of contractile markers (α-SMA, calponin, SM22α, and smoothelin) in VSMCs. While silencing miR-491-5p promoted viability and migration, and significantly suppressed the expression of α-SMA, calponin, SM22α, and smoothelin. Overall, miR-491-5p targeted UBE2G2, SLC16A3, and PNO1 and regulated the dysfunctions in VSMCs.

Keyword

Coronary artery disease; miR-491-5p; PNO1; SLC16A3; UBE2G2

Figure

  • Fig. 1 Expression of 10 mRNAs in plaques and adjacent intimal tissues from atherosclerosis (AS) patients. (A) Expression levels of ten mRNAs (UBE2G2, SNORD45C, SNORD45A, SNORD45B, RABGGTB, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) in plaques and adjacent intima from coronary AS patients were measured using RT-qPCR with pared t-test, as presented by the scatter diagram. (B) mRNAs (UBE2G2, SNORD45C, SNORD45A, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) with significant difference were listed in the bar graph. *p < 0.05, **p < 0.01, NS indicates no significance.

  • Fig. 2 Expression of 10 mRNAs in serum from atherosclerosis (AS) patients and healthy controls. (A) Expression levels of ten mRNAs (UBE2G2, SNORD45C, SNORD45A, SNORD45B, RABGGTB, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) in the serum of coronary AS patients and healthy patients were measured using RT-qPCR with unpaired t-test. (B) mRNAs (UBE2G2, SNORD45C, SNORD45A, SLC16A3, POLR2C, PNO1, CEMP1, AMDHD2) with significant difference were listed. *p < 0.05, **p < 0.01, ***p < 0.001, NS indicates no significance.

  • Fig. 3 Potential upstream miRNAs for the abnormally expressed mRNAs. (A) TargetScan predicted the potential upstream miRNAs of UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2. (B) Expression levels of 11 miRNAs in plaques and adjacent intima from coronary AS patients were examined using RT-qPCR with pared t-test. (C) Expression levels of 11 miRNAs in the serum of coronary atherosclerosis (AS) patients and healthy patients were examined using RT-qPCR with unpaired t-test. *p < 0.05, **p < 0.01.

  • Fig. 4 MiR-491-5p negatively regulates UBE2G2, SLC16A3, PNO1 expression in vascular smooth muscle cells (VSMCs). (A) Overexpression efficiency of miR-491-5p in VSMCs was evaluated using RT-qPCR. (B) Expression levels of UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2 in VSMCs transfected with miR-491-5p mimics and NC mimics were examined by RT-qPCR. (C) Protein levels of UBE2G2, SLC16A3, POLR2C, PNO1, and AMDHD2 in VSMCs transfected with miR-491-5p mimics and NC mimics were examined by Western blot. (D) RNA pull down assay was for detecting whether UBE2G2, SLC16A3, and PNO1 bind with miR-491-5p. *p < 0.05, **p < 0.01, ***p < 0.001.

  • Fig. 5 MiR-491-5p inhibited viability, migration, and increased expression of contractile markers in vascular smooth muscle cells (VSMCs). (A) Cell counting kit 8 (CCK-8) revealed the viability in VSMCs with transfection of miR-491-5p mimics and NC mimics. (B) Wound healing was performed to detect the migration capacity of VSMCs with transfection of miR-491-5p mimics and NC mimics. (C) The protein levels of contractile markers (α-SMA, calponin, SM22α, and smoothelin) in VSMCs with transfection of miR-491-5p mimics and NC mimics were measured by Western blot. *p < 0.05, **p < 0.01.

  • Fig. 6 MiR-491-5p antagomir promotes viability, migration, and increased expression of contractile markers in vascular smooth muscle cells (VSMCs). (A) CCK-8 revealed the viability in VSMCs with transfection of NC antagomir, miR-491-5p antagomir, miR-491-5p antagomir+sh-NC, miR-491-5p antagomir+sh-UBE2G2, miR-491-5p antagomir+sh-SLC16A3, and miR-491-5p antagomir+sh PNO1. (B) Wound healing was performed to detect the migration capacity of VSMCs with transfection of NC antagomir, miR-491-5p antagomir, miR-491-5p antagomir+sh-NC, miR-491-5p antagomir+sh-UBE2G2, miR-491-5p antagomir+sh- SLC16A3, and miR-491-5p antagomir+sh PNO1. (C) The protein levels of contractile markers (α-SMA, calponin, SM22α, and smoothelin) in VSMCs with transfection of NC antagomir, miR-491-5p antagomir, miR-491-5p antagomir+sh-NC, miR-491-5p antagomir+sh-UBE2G2, miR-491-5p antagomir+sh- SLC16A3, and miR-491-5p antagomir+sh PNO1. *p < 0.05, **p < 0.01.


Reference

1. Ou H, Liu C, Feng W, Xiao X, Tang S, Mo Z. 2018; Role of AMPK in atherosclerosis via autophagy regulation. Sci China Life Sci. 61:1212–1221. DOI: 10.1007/s11427-017-9240-2. PMID: 29656339.
Article
2. Boudoulas KD, Triposciadis F, Geleris P, Boudoulas H. 2016; Coronary atherosclerosis: pathophysiologic basis for diagnosis and management. Prog Cardiovasc Dis. 58:676–692. DOI: 10.1016/j.pcad.2016.04.003. PMID: 27091673.
Article
3. Bennett MR, Sinha S, Owens GK. 2016; Vascular smooth muscle cells in atherosclerosis. Circ Res. 118:692–702. DOI: 10.1161/CIRCRESAHA.115.306361. PMID: 26892967. PMCID: PMC4762053.
Article
4. Wang J, Uryga AK, Reinhold J, Figg N, Baker L, Finigan A, Gray K, Kumar S, Clarke M, Bennett M. 2015; Vascular smooth muscle cell senescence promotes atherosclerosis and features of plaque vulnerability. Circulation. 132:1909–1919. DOI: 10.1161/CIRCULATIONAHA.115.016457. PMID: 26416809.
Article
5. Johnson JL. 2014; Emerging regulators of vascular smooth muscle cell function in the development and progression of atherosclerosis. Cardiovasc Res. 103:452–460. DOI: 10.1093/cvr/cvu171. PMID: 25053639.
Article
6. Hutcheson JD, Goettsch C, Bertazzo S, Maldonado N, Ruiz JL, Goh W, Yabusaki K, Faits T, Bouten C, Franck G, Quillard T, Libby P, Aikawa M, Weinbaum S, Aikawa E. 2016; Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques. Nat Mater. 15:335–343. DOI: 10.1038/nmat4519. PMID: 26752654. PMCID: PMC4767675.
Article
7. Jiang XI, Luo Y, Zhao S, Chen Q, Jiang C, Dai Y, Chen Y, Cao Z. 2015; Clinical significance and expression of microRNA in diabetic patients with erectile dysfunction. Exp Ther Med. 10:213–218. DOI: 10.3892/etm.2015.2443. PMID: 26170937. PMCID: PMC4486873.
Article
8. Zhu N, Zhang D, Chen S, Liu X, Lin L, Huang X, Guo Z, Liu J, Wang Y, Yuan W, Qin Y. 2011; Endothelial enriched microRNAs regulate angiotensin II-induced endothelial inflammation and migration. Atherosclerosis. 215:286–293. DOI: 10.1016/j.atherosclerosis.2010.12.024. PMID: 21310411.
Article
9. Ambros V. 2004; The functions of animal microRNAs. Nature. 431:350–355. DOI: 10.1038/nature02871. PMID: 15372042.
Article
10. Ndrepepa G, Colleran R, Kastrati A. 2018; Gamma-glutamyl transferase and the risk of atherosclerosis and coronary heart disease. Clin Chim Acta. 476:130–138. DOI: 10.1016/j.cca.2017.11.026. PMID: 29175647.
Article
11. Xu Z, Han Y, Liu J, Jiang F, Hu H, Wang Y, Liu Q, Gong Y, Li X. 2015; miR-135b-5p and miR-499a-3p promote cell proliferation and migration in atherosclerosis by directly targeting MEF2C. Sci Rep. 5:12276. DOI: 10.1038/srep12276. PMID: 26184978. PMCID: PMC4505325.
Article
12. Zhang R, Sui L, Hong X, Yang M, Li W. 2017; miR-448 promotes vascular smooth muscle cell proliferation and migration in through directly targeting MEF2C. Environ Sci Pollut Res Int. 24:22294–22300. DOI: 10.1007/s11356-017-9771-1. PMID: 28799067.
Article
13. Lin B, Feng DG, Wang F, Wang JX, Xu CG, Zhao H, Cheng ZY. 2016; miR-365 participates in coronary atherosclerosis through regulating IL-6. Eur Rev Med Pharmacol Sci. 20:5186–5192. PMID: 28051250.
14. Lu L, Cai M, Peng M, Wang F, Zhai X. 2019; miR-491-5p functions as a tumor suppressor by targeting IGF2 in colorectal cancer. Cancer Manag Res. 11:1805–1816. DOI: 10.2147/CMAR.S183085. PMID: 30863186. PMCID: PMC6391127.
15. Guo J, Luo C, Yang Y, Dong J, Guo Z, Yang J, Lian H, Ye C, Liu M. 2021; miR-491-5p, as a tumor suppressor, prevents migration and invasion of breast cancer by targeting ZNF-703 to regulate AKT/mTOR pathway. Cancer Manag Res. 13:403–413. DOI: 10.2147/CMAR.S279747. PMID: 33488122. PMCID: PMC7816048.
Article
16. Sun R, Liu Z, Tong D, Yang Y, Guo B, Wang X, Zhao L, Huang C. 2017; miR-491-5p, mediated by Foxi1, functions as a tumor suppressor by targeting Wnt3a/β-catenin signaling in the development of gastric cancer. Cell Death Dis. 8:e2714. DOI: 10.1038/cddis.2017.134. PMID: 28358374. PMCID: PMC5386537.
Article
17. He Z, Wang Y, He Q, Chen M. 2020; microRNA-491-5p protects against atherosclerosis by targeting matrix metallopeptidase-9. Open Med (Wars). 15:492–500. DOI: 10.1515/med-2020-0047. PMID: 33313408. PMCID: PMC7706122.
Article
18. Yuan M, Zhan Q, Duan X, Song B, Zeng S, Chen X, Yang Q, Xia J. 2013; A functional polymorphism at miR-491-5p binding site in the 3'-UTR of MMP-9 gene confers increased risk for atherosclerotic cerebral infarction in a Chinese population. Atherosclerosis. 226:447–452. DOI: 10.1016/j.atherosclerosis.2012.11.026. PMID: 23257658.
Article
19. Woo CC, Liu W, Lin XY, Dorajoo R, Lee KW, Richards AM, Lee CN, Wongsurawat T, Nookaew I, Sorokin V. 2019; The interaction between 30b-5p miRNA and MBNL1 mRNA is involved in vascular smooth muscle cell differentiation in patients with coronary atherosclerosis. Int J Mol Sci. 21:11. DOI: 10.3390/ijms21010011. PMID: 31861407. PMCID: PMC6982107.
Article
20. Bartel DP. 2004; MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281–297. DOI: 10.1016/S0092-8674(04)00045-5. PMID: 14744438.
21. Song Z, Li G. 2010; Role of specific microRNAs in regulation of vascular smooth muscle cell differentiation and the response to injury. J Cardiovasc Transl Res. 3:246–250. DOI: 10.1007/s12265-010-9163-0. PMID: 20543900. PMCID: PMC2883267.
Article
22. Chen Q, Yang F, Guo M, Wen G, Zhang C, Luong le A, Zhu J, Xiao Q, Zhang L. 2015; miRNA-34a reduces neointima formation through inhibiting smooth muscle cell proliferation and migration. J Mol Cell Cardiol. 89(Pt A):75–86. DOI: 10.1016/j.yjmcc.2015.10.017. PMID: 26493107.
Article
23. Davis-Dusenbery BN, Wu C, Hata A. 2011; Micromanaging vascular smooth muscle cell differentiation and phenotypic modulation. Arterioscler Thromb Vasc Biol. 31:2370–2377. DOI: 10.1161/ATVBAHA.111.226670. PMID: 22011749. PMCID: PMC4429757.
Article
24. Albinsson S, Sessa WC. 2011; Can microRNAs control vascular smooth muscle phenotypic modulation and the response to injury? Physiol Genomics. 43:529–533. DOI: 10.1152/physiolgenomics.00146.2010. PMID: 20841497. PMCID: PMC3110893.
Article
25. Tabas I. 2010; Macrophage death and defective inflammation resolution in atherosclerosis. Nat Rev Immunol. 10:36–46. DOI: 10.1038/nri2675. PMID: 19960040. PMCID: PMC2854623.
Article
26. Libby P, Ridker PM, Hansson GK. 2011; Progress and challenges in translating the biology of atherosclerosis. Nature. 473:317–325. DOI: 10.1038/nature10146. PMID: 21593864.
Article
27. Pordzik J, Pisarz K, De Rosa S, Jones AD, Eyileten C, Indolfi C, Malek L, Postula M. 2018; The potential role of platelet-related microRNAs in the development of cardiovascular events in high-risk populations, including diabetic patients: a review. Front Endocrinol (Lausanne). 9:74. DOI: 10.3389/fendo.2018.00074. PMID: 29615970. PMCID: PMC5869202.
Article
28. Fasolo F, Di Gregoli K, Maegdefessel L, Johnson JL. 2019; Non-coding RNAs in cardiovascular cell biology and atherosclerosis. Cardiovasc Res. 115:1732–1756. DOI: 10.1093/cvr/cvz203. PMID: 31389987. PMCID: PMC7967706.
Article
29. Hueso M, Mallén A, Casas Á, Guiteras J, Sbraga F, Blasco-Lucas A, Lloberas N, Torras J, Cruzado JM, Navarro E. 2020; Integrated miRNA/mRNA counter-expression analysis highlights oxidative stress-related genes CCR7 and FOXO1 as blood markers of coronary arterial disease. Int J Mol Sci. 21:1943. DOI: 10.3390/ijms21061943. PMID: 32178422. PMCID: PMC7139611.
Article
30. He Y, Lin L, Cao J, Mao X, Qu Y, Xi B. 2015; Up-regulated miR-93 contributes to coronary atherosclerosis pathogenesis through targeting ABCA1. Int J Clin Exp Med. 8:674–681. PMID: 25785043. PMCID: PMC4358498.
31. Fan JL, Zhang L, Bo XH. 2020; miR-126 on mice with coronary artery disease by targeting S1PR2. Eur Rev Med Pharmacol Sci. 24:893–904. DOI: 10.26355/eurrev_202001_20074. PMID: 32016996.
Full Text Links
  • KJPP
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr