Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

MicroRNA-17-3p suppresses NF-κB-mediated endothelial inflammation by targeting NIK and IKKβ binding protein

Abstract

Nuclear factor kappa B (NF-κB) activation contributes to many vascular inflammatory diseases. The present study tested the hypothesis that microRNA-17-3p (miR-17-3p) suppresses the pro-inflammatory responses via NF-κB signaling in vascular endothelium. Human umbilical vein endothelial cells (HUVECs), transfected with or without miR-17-3p agomir/antagomir, were exposed to lipopolysaccharide (LPS), and the inflammatory responses were determined. The cellular target of miR-17-3p was examined with dual-luciferase reporter assay. Mice were treated with miR-17-3p agomir and the degree of LPS-induced inflammation was determined. In HUVECs, LPS caused upregulation of miR-17-3p. Overexpression of miR-17-3p in HUVECs inhibited NIK and IKKβ binding protein (NIBP) protein expression and suppressed LPS-induced phosphorylation of inhibitor of kappa Bα (IκBα) and NF-κB-p65. The reduced NF-κB activity was paralleled by decreased protein levels of NF-κB-target gene products including pro-inflammatory cytokine [interleukin 6], chemokines [interleukin 8 and monocyte chemoattractant protein-1] and adhesion molecules [vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin]. Immunostaining revealed that overexpression of miR-17-3p reduced monocyte adhesion to LPS-stimulated endothelial cells. Inhibition of miR-17-3p with antagomir has the opposite effect on LPS-induced inflammatory responses in HUVECs. The anti-inflammatory effect of miR-17-3p was mimicked by NIBP knockdown. In mice treated with LPS, miR-17-3p expression was significantly increased. Systemic administration of miR-17-3p for 3 days suppressed LPS-induced NF-κB activation and monocyte adhesion to endothelium in lung tissues of the mice. In conclusion, miR-17-3p inhibits LPS-induced NF-κB activation in HUVECs by targeting NIBP. The findings therefore suggest that miR-17-3p is a potential therapeutic target/agent in the management of vascular inflammatory diseases.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: miR-17-3p suppresses LPS-induced pro-inflammatory cytokines and adhesion molecules in endothelial cells.
Fig. 2: miR-17-3p inhibits monocytes adhesion to activated endothelial cell monolayers.
Fig. 3: miR-17-3p inhibits phosphorylation of IκBα and p65 in endothelial cells.
Fig. 4: miR-17-3p directly targets expression of NIBP protein in endothelial cells.
Fig. 5: miR-17-3p suppresses LPS-induced NF-κB activation and expression of adhesion molecules in mice.
Fig. 6: Schematic summary.

References

  1. 1.

    Schöffel U, Kopp KH, Männer H, Vogel F, Mittermayer C. Human endothelial cell proliferation inhibiting activity in the sera of patients suffering from ‘shock’ or ‘sepsis’. Eur J Clin Invest. 1982;12:165–71.

    PubMed  Article  Google Scholar 

  2. 2.

    McKenna TM, Martin FM, Chernow B, Briglia FA. Vascular endothelium contributes to decreased aortic contractility in experimental sepsis. Circ Shock. 1986;19:267–73.

    CAS  PubMed  Google Scholar 

  3. 3.

    Constantinides P. Importance of the endothelium and blood platelets in the pathogenesis of atherosclerosis. Triangle. 1976;15:53–61.

    CAS  PubMed  Google Scholar 

  4. 4.

    Landmesser U, Hornig B, Drexler H. Endothelial function: a critical determinant in atherosclerosis? Circulation. 2004;109:II27–33.

    PubMed  Article  Google Scholar 

  5. 5.

    Lusis AJ. Atherosclerosis. Nature. 2000;407:233–41.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Libby P. Inflammation in atherosclerosis. Arterioscler Thromb Vasc Biol. 2012;32:2045–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Ince C, Mayeux PR, Nguyen T, Gomez H, Kellum JA, Ospina-Tascón GA, et al. The endothelium in sepsis. Shock. 2016;45:259–70.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Libby P. Inflammatory mechanisms: the molecular basis of inflammation and disease. Nutr Rev. 2007;65:S140–46.

    PubMed  Article  Google Scholar 

  9. 9.

    Gareus R, Kotsaki E, Xanthoulea S, van der Made I, Gijbels MJ, Kardakaris R, et al. Endothelial cell-specific NF-kappaB inhibition protects mice from atherosclerosis. Cell Metab. 2008;8:372–83.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Sun X, Icli B, Wara AK, Belkin N, He S, Kobzik L, et al. MicroRNA-181b regulates NF-κB-mediated vascular inflammation. J Clin Invest. 2012;122:1973–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Xanthoulea S, Curfs DM, Hofker MH, de Winther MP. Nuclear factor kappa B signaling in macrophage function and atherogenesis. Curr Opin Lipido. 2005;16:536–42.

    CAS  Article  Google Scholar 

  12. 12.

    Cai Y, Sukhova GK, Wong HK, Xu A, Tergaonkar V, Vanhoutte PM, et al. Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling and is highly expressed in human atherosclerotic lesions. Cell Cycle. 2015;14:3580–92.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Ghosh S, May MJ, Kopp EB. NF-κB and rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol. 1998;16:225–60.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Israël A. The IKK complex, a central regulator of NF-κB activation. Cold Spring Harb Perspect Biol. 2010;2:a000158.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  15. 15.

    Xiao G, Harhaj EW, Sun SC. NF-kappaB-inducing kinase regulates the processing of NF-kappaB2 p100. Mol Cell. 2001;7:401–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. 16.

    Senftleben U, Cao Y, Xiao G, Greten FR, Krähn G, Bonizzi G, et al. Activation by IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway. Science. 2001;293:1495–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Ramakrishnan P, Wang W, Wallach D. Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase. Immunity. 2004;21:477–89.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Sawa Y, Ueki T, Hata M, Iwasawa K, Tsuruga E, Kojima H, et al. LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium. J Histochem Cytochem. 2008;56:97–109.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature. 2008;455:64–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Lam JK, Chow MY, Zhang Y, Leung SW. siRNA versus miRNA as therapeutics for gene silencing. Mol Ther Nucleic Acids. 2015;4:e252.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Wang Q, Li YC, Wang J, Kong J, Qi Y, Quigg RJ, et al. miR-17-92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proc Natl Acad Sci USA. 2008;105:2889–94.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. 23.

    Danielson LS, Park DS, Rotllan N, Chamorro-Jorganes A, Guijarro MV, Fernandez-Hernando C, et al. Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis. FASEB J. 2013;27:1460–67.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL. Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol. 2007;310:442–53.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Shi J, Bei Y, Kong X, Liu X, Lei Z, Xu T, et al. miR-17-3p contributes to exercise-induced cardiac growth and protects against myocardial ischemia-reperfusion injury. Theranostics. 2017;7:664–76.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Tian B, Maidana DE, Dib B, Miller JB, Bouzika P, Miller JW, et al. miR-17-3p exacerbates oxidative damage in human retinal pigment epithelial cells. PLoS ONE. 2016;11:e0160887.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. 27.

    Lu D, Tang L, Zhuang Y, Zhao P. miR-17-3P regulates the proliferation and survival of colon cancer cells by targeting Par4. Mol Med Rep. 2018;17:618–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Suárez Y, Wang C, Manes TD, Pober JS. Cutting edge: TNF-induced microRNAs regulate TNF-induced expression of E-selectin and intercellular adhesion molecule-1 on human endothelial cells: feedback control of inflammation. J Immunol. 2010;184:21–5.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  29. 29.

    Yang F, Tang E, Guan K, Wang CY. IKKβ plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J Immunol. 2003;170:5630–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Kremmidiotis G, Gardner AE, Settasatian C, Savoia A, Sutherland GR, Callen DF. Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein. Genomics. 2001;76:58–65.

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Hu WH, Pendergast JS, Mo XM, Brambilla R, Bracchi-Ricard V, Li F, et al. NIBP, a novel NIK and IKK(beta)-binding protein that enhances NF-(kappa)B activation. J Biol Chem. 2005;280:29233–41.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Copeland S, Warren HS, Lowry SF, Calvano SE, Remick D. Inflammation and the host response to injury investigators. Acute inflammatory response to endotoxin in mice and humans. Clin Diagn Lab Immunol. 2005;12:60–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood. 2003;101:3765–77.

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006;6:508–19.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell. 2009;136:642–55.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Finnegan EF, Pasquinelli AE. MicroRNA biogenesis: regulating the regulators. Crit Rev Biochem Mol Biol. 2013;48:51–68.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Rawal S, Manning P, Katare R. Cardiovascular microRNAs: as modulators and diagnostic biomarkers of diabetic heart disease. Cardiovasc Diabetol. 2014;13:44.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  38. 38.

    Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003;115:209–16.

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Schwarz DS, Hutvágner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell. 2003;115:199–208.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Okamura K, Phillips MD, Tyler DM, Duan H, Chou YT, Lai EC. The regulatory activity of microRNA* species has substantial influence on microRNA and 3′ UTR evolution. Nat Struct Mol Biol. 2008;15:354–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Winter J, Diederichs S. Argonaute-3 activates the let-7a passenger strand microRNA. RNA Biol. 2013;10:1631–43.

    PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Mir A, Kaufman L, Noor A, Motazacker MM, Jamil T, Azam M, et al. Identification of mutations in TRAPPC9, which encodes the NIK- and IKK-beta-binding protein, in nonsyndromic autosomal-recessive mental retardation. Am J Hum Genet. 2009;85:909–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Zhang Y, Bitner D, Pontes Filho AA, Li F, Liu S, Wang H, et al. Expression and function of NIK- and IKK2-binding protein (NIBP) in mouse enteric nervous system. Neurogastroenterol Motil. 2014;26:77–97.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    Zhang Y, Liu S, Wang H, Yang W, Li F, Yang F, et al. Elevated NIBP/TRAPPC9 mediates tumorigenesis of cancer cells through NFκB signaling. Oncotarget. 2015;6:6160–78.

    PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Brennecke J, Stark A, Russell RB, Cohen SM. Principles of microRNA-target recognition. PLoS Biol. 2005;3:e85.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46.

    Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007;27:91–105.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Nielsen CB, Shomron N, Sandberg R, Hornstein E, Kitzman J, Burge CB. Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. RNA. 2007;13:1894–910.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature. 2008;455:58–63.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Wu XY, Fan WD, Fang R, Wu GF. Regulation of microRNA-155 in endothelial inflammation by targeting nuclear factor (NF)-κB P65. J Cell Biochem. 2014;115:1928–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Cheng HS, Sivachandran N, Lau A, Boudreau E, Zhao JL, Baltimore D, et al. MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways. EMBO Mol Med. 2013;5:1017–34.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  51. 51.

    Sun X, He S, Wara AKM, Icli B, Shvartz E, Tesmenitsky Y, et al. Systemic delivery of microRNA-181b inhibits nuclear factor-κB activation, vascular inflammation, and atherosclerosis in apolipoprotein E-deficient mice. Circ Res. 2014;114:32–40.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Sun X, Sit A, Feinberg MW. Role of miR-181 family in regulating vascular inflammation and immunity. Trends Cardiovasc Med. 2014;24:105–12.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Yan H, Song K, Zhang G. MicroRNA-17-3p promotes keratinocyte cells growth and metastasis via targeting MYOT and regulating Notch1/NF-κB pathways. Pharmazie. 2017;72:543–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Frangogiannis NG. Targeting the inflammatory response in healing myocardial infarcts. Curr Med Chem. 2006;13:1877–93.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    Kim JW, Jin YC, Kim YM, Rhie S, Kim HJ, Seo HG, et al. Daidzein administration in vivo reduces myocardial injury in a rat ischemia/reperfusion model by inhibiting NF-kappaB activation. Life Sci. 2009;84:227–34.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. 56.

    Yuan T, Yang Z, Xian S, Chen Y, Wang L, Chen W, et al. Dexmedetomidine-mediated regulation of miR-17-3p in H9C2 cells after hypoxia/reoxygenation injury. Exp Ther Med. 2020;20:917–25.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Bohl TE, Aihara H. Current Progress in the structural and biochemical characterization of proteins involved in the assembly of lipopolysaccharide. Int J Microbiol. 2018;2018:5319146.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  58. 58.

    Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine. 2008;42:145–51.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Health and Medical Research Fund (16151212) of the Food and Health Bureau of the Government of the Hong Kong Special Administrative Region (to SWSL), a Seed Fund for Basic Research of the University of Hong Kong (to SWSL), and the National Institutes of Health (HL115141, HL117994, HL134849, and GM115605 to MWF), the Arthur K. Watson Charitable Trust (to MWF), and the Dr. Ralph & Marian Falk Medical Research Trust (to MWF).

Author information

Affiliations

Authors

Contributions

YC, YZ, HC, and XHS conceived and designed the study, performed the experiments, analyzed the data and wrote the manuscript. PZ, LZ, MYL, and FZ performed some experiments and analyzed the data. ZYX participated in the experiment design and the interpretation of results. RYKM, MWF, and SWSL designed the experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Susan Wai-Sum Leung.

Ethics declarations

Competing interests

The authors declare no competing interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cai, Y., Zhang, Y., Chen, H. et al. MicroRNA-17-3p suppresses NF-κB-mediated endothelial inflammation by targeting NIK and IKKβ binding protein. Acta Pharmacol Sin (2021). https://doi.org/10.1038/s41401-021-00611-w

Download citation

Keywords

  • endothelial cells
  • inflammation
  • miR-17-3p
  • NIK and IKKβ binding protein
  • nuclear factor kappa B

Search

Quick links