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Intronic microRNA suppresses endothelial nitric oxide synthase expression and endothelial cell proliferation via inhibition of STAT3 signaling

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Abstract

Intronic microRNA (miRNAs) suppressed the expression of endothelial nitric oxide synthase (eNOS) gene in endothelial cells (ECs). This study was to investigate the role of signal transducer and activator of transcription 3 (STAT3) in the regulation of eNOS expression and vascular EC proliferation by the intronic 27-nucleotide (nt) miRNA derived from the 27-base pair repeats in intron 4 of eNOS gene. A detectable level of the 27-nt miRNA was present in the control ECs. Overexpression of the 27-nt miRNA dramatically suppressed the expression of eNOS and STAT3 at both transcription and translation levels in ECs in association with significant inhibition of EC proliferation. Mutation of the 27-nt miRNA at the 3′-terminal region resulted in substantial reduction of the inhibitory effect of miRNA on eNOS and STAT3 expression, and EC proliferation. Overexpression of active STAT3 significantly reversed the inhibitory effect of the 27-nt miRNA on eNOS expression and EC proliferation. In summary, we demonstrated that the 27-nt intronic miRNA functioned as a negative regulator for the expression of its host gene eNOS and cell proliferation in ECs. The sequence in 3′-terminal region played a key role in the function of the 27-nt miRNA. The regulatory effect of the intronic miRNA on eNOS gene expression was associated with miRNA polymorphisms, and mediated through inhibition of STAT3 signaling in ECs.

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References

  1. Lai EC (2003) MicroRNAs: runts of the genome assert themselves. Curr Biol 13:R925–R936

    Article  PubMed  CAS  Google Scholar 

  2. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

    Article  PubMed  CAS  Google Scholar 

  3. http://microrna.sanger.ac.uk/

  4. Abrahante E, Daul AL, Li M, Volk ML, Tennessen JM, Miller EA, Rougvie AE (2003) The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs. Dev Cell 4:625–637

    Article  PubMed  CAS  Google Scholar 

  5. Gandikota M, Birkenbihl RP, Höhmann S, Cardon GH, Saedler H, Huijser P (2007) The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J 49:683–693

    Article  PubMed  CAS  Google Scholar 

  6. Liu X, Cheng Y, Zhang S, Lin Y, Yang J, Zhang C (2009) A necessary role of miR-221 and miR-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia. Circ Res 104:476–487

    Article  PubMed  CAS  Google Scholar 

  7. Asada S, Takahashi T, Isodono K, Adachi A, Imoto H, Ogata T, Ueyama T, Matsubara H, Oh H (2008) Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells to apoptosis. Am J Physiol Heart Circ Physiol 295(6):H2512–H2521

    Article  PubMed  CAS  Google Scholar 

  8. Xu P, Vernooy SY, Guo M, Hay BA (2003) The Drosophila microRNA mir-14 suppresses cell death and is required for normal fat metabolism. Curr Biol 13:790–795

    Article  PubMed  CAS  Google Scholar 

  9. Thum T, Gross C, Fiedler J, Fischer T, Kissler S, Bussen M, Galuppo P, Just S, Rottbauer W, Frantz S, Castoldi M, Soutschek J, Koteliansky V, Rosenwald A, Basson MA, Licht JD, Pena JT, Rouhanifard SH, Muckenthaler MU, Tuschl T, Martin GR, Bauersachs J, Engelhardt S (2008) MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature 456:980–984

    Article  PubMed  CAS  Google Scholar 

  10. Lovis P, Roggli E, Laybutt DR, Gattesco S, Yang JY, Widmann C, Abderrahmani A, Regazzi R (2008) Alterations in microRNA expression contribute to fatty acid-induced pancreatic beta-cell dysfunction. Diabetes 5710:2728–2736

    Article  Google Scholar 

  11. Noonan EJ, Place RF, Pookot D, Basak S, Whitson JM, Hirata H, Giardina C, Dahiya R (2009) miR-449a targets HDAC-1 and induces growth arrest in prostate cancer. Oncogene 28:1714–1724

    Article  PubMed  CAS  Google Scholar 

  12. Rodriguez A, Griffiths-Jones S, Ashurst JL, Bradley A (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res 14:1902–1910

    Article  PubMed  CAS  Google Scholar 

  13. Ying SY, Lin SL (2004) Intron-derived microRNAs-fine tuning of gene functions. Gene 342:25–28

    Article  PubMed  CAS  Google Scholar 

  14. Lin SL, Chang D, Wu DY, Ying SY (2003) A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution. Biochem Biophys Res Commun 310:754–760

    Article  PubMed  CAS  Google Scholar 

  15. Clement JQ, Qian L, Kaplinsky N, Wilkinson MF (1999) The stability and fate of a spliced intron from vertebrate cells. RNA 5:206–220

    Article  PubMed  CAS  Google Scholar 

  16. Saito Y, Friedman JM, Chihara Y, Egger G, Chuang JC, Liang G (2009) Epigenetic therapy upregulates the tumor suppressor microRNA-126 and its host gene EGFL7 in human cancer cells. Biochem Biophys Res Commun 379:726–731

    Article  PubMed  CAS  Google Scholar 

  17. Li XM, Dong XP, Luo SW, Zhang B, Lee DH, Ting AK, Neiswender H, Kim CH, Carpenter-Hyland E, Gao TM, Xiong WC, Mei L (2008) Retrograde regulation of motoneuron differentiation by muscle beta-catenin. Nat Neurosci 11:262–268

    Article  PubMed  CAS  Google Scholar 

  18. Tai SC, Robb GB, Marsden PA (2004) Endothelial nitric oxide synthase: a new paradigm for gene regulation in the injured blood vessel. Arterioscler Thromb Vasc Biol 24:405–412

    Article  PubMed  CAS  Google Scholar 

  19. Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, Shi XM, Tsui LC, Schappert KT (1993) Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 268:17478–17488

    PubMed  CAS  Google Scholar 

  20. Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, Nakao K (1999) T-786→C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation 99:2864–2870

    PubMed  CAS  Google Scholar 

  21. Kim IJ, Bae J, Lim SW, Cha DH, Cho HJ, Kim S, Yang DH, Hwang SG, Oh D, Kim NK (2007) Influence of endothelial nitric oxide synthase gene polymorphisms (-786T>C, 4a4b, 894G>T) in Korean patients with coronary artery disease. Thromb Res 119:579–585

    Article  PubMed  CAS  Google Scholar 

  22. Spoto B, Benedetto FA, Testa A, Tripepi G, Mallamaci F, Maas R, Boeger RH, Zoccali C (2007) An additive effect of endothelial nitric oxide synthase gene polymorphisms contributes to the severity of atherosclerosis in patients on dialysis. Am J Hypertens 20:758–763

    Article  PubMed  CAS  Google Scholar 

  23. Rittig K, Holder K, Stock J, Tschritter O, Peter A, Stefan N, Fritsche A, Machicao F, Häring HU, Balletshofer B (2008) Endothelial NO-synthase intron 4 polymorphism is associated with disturbed in vivo nitric oxide production in individuals prone to type II diabetes. Horm Metab Res 40:13–17

    Article  PubMed  CAS  Google Scholar 

  24. Wang XL, Sim AS, Badenhop RF, McCredie RM, Wilcken DE (1996) A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med 2:41–45

    Article  PubMed  CAS  Google Scholar 

  25. Wattanapitayakul SK, Mihm MJ, Young AP, Bauer JA (2001) Therapeutic implications of human endothelial nitric oxide synthase gene polymorphism. Trends Pharmacol Sci 22:361–368

    Article  PubMed  CAS  Google Scholar 

  26. Wang J, Dudley D, Wang XL (2002) Haplotype-specific effects on endothelial NO synthase promoter efficiency: modifiable by cigarette smoking. Arterioscler Thromb Vasc Biol 22:e1–e4

    Article  PubMed  Google Scholar 

  27. Potenza MA, Gagliardi S, Nacci C, Carratu’ MR, Montagnani M (2009) Endothelial dysfunction in diabetes: from mechanisms to therapeutic targets. Curr Med Chem 16:94–112

    Article  PubMed  CAS  Google Scholar 

  28. Nikitenko LL (2009) Vascular endothelium in cancer. Cell Tissue Res 335:223–240

    Article  PubMed  Google Scholar 

  29. Higashi Y, Noma K, Yoshizumi M, Kihara Y (2009) Endothelial function and oxidative stress in cardiovascular diseases. Circ J 73:411–418

    Article  PubMed  CAS  Google Scholar 

  30. Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F (1994) Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 94:2036–2044

    Article  PubMed  CAS  Google Scholar 

  31. Zhang MX, Zhang C, Shen YH, Wang J, Li XN, Zhang Y, Coselli J, Wang XL (2008) Biogenesis of short intronic repeat 27-nucleotide small RNA from endothelial nitric-oxide synthase gene. J Biol Chem 283:14685–14693

    Article  PubMed  CAS  Google Scholar 

  32. Zhang MX, Ou H, Shen YH, Wang J, Wang J, Coselli J, Wang XL (2005) Regulation of endothelial nitric oxide synthase by small RNA. Proc Natl Acad Sci USA 102:16967–16972

    Article  PubMed  CAS  Google Scholar 

  33. Ou H, Shen YH, Utama B, Wang J, Wang X, Coselli J, Wang XL (2005) Effect of nuclear actin on endothelial nitric oxide synthase expression. Arterioscler Thromb Vasc Biol 25:2509–2514

    Article  PubMed  CAS  Google Scholar 

  34. Chen CZ, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303:83–86

    Article  PubMed  CAS  Google Scholar 

  35. Liu W, Tang F, Deng Y, Li X, Lan T, Zhang X, Huang H, Liu P (2009) Berberine reduces fibronectin and collagen accumulation in rat glomerular mesangial cells cultured under high glucose condition. Mol Cell Biochem 325:99–105

    Article  PubMed  CAS  Google Scholar 

  36. Chau MN, El Touny LH, Jagadeesh S, Banerjee PP (2007) Physiologically achievable concentrations of genistein enhance telomerase activity in prostate cancer cells via the activation of STAT3. Carcinogenesis 28:2282–2290

    Article  PubMed  CAS  Google Scholar 

  37. Namba T, Koike H, Murakami K, Aoki M, Makino H, Hashiya N, Ogihara T, Kaneda Y, Kohno M, Morishita R (2003) Angiogenesis induced by endothelial nitric oxide synthase gene through vascular endothelial growth factor expression in a rat hindlimb ischemia model. Circulation 108:2250–2257

    Article  PubMed  CAS  Google Scholar 

  38. Dudzinski DM, Michel T (2007) Life history of eNOS: partners and pathways. Cardiovasc Res 75:247–260

    Article  PubMed  CAS  Google Scholar 

  39. Barik S (2008) An intronic microRNA silences genes that are functionally antagonistic to its host gene. Nucleic Acids Res 36:5232–5241

    Article  PubMed  CAS  Google Scholar 

  40. Ronchetti D, Lionetti M, Mosca L, Agnelli L, Andronache A, Fabris S, Deliliers GL, Neri A (2008) An integrative genomic approach reveals coordinated expression of intronic miR-335, miR-342, and miR-561 with deregulated host genes in multiple myeloma. BMC Med Genomics 1:37

    Article  PubMed  Google Scholar 

  41. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of mammalian microRNA targets. Cell 115:787–798

    Article  PubMed  CAS  Google Scholar 

  42. Clanton-Arrowood K, McGurk J, Schroeder SJ (2008) 3′ terminal nucleotides determine thermodynamic stabilities of mismatches at the ends of RNA helices. Biochemistry 47:13418–13427

    Article  PubMed  CAS  Google Scholar 

  43. Sethupathy P, Collins FS (2008) MicroRNA target site polymorphisms and human disease. Trends Genet 24:489–497

    Article  PubMed  CAS  Google Scholar 

  44. Hu Z, Chen J, Tian T, Zhou X, Gu H, Xu L, Zeng Y, Miao R, Jin G, Ma H, Chen Y, Shen H (2008) Genetic variants of miRNA sequences and non-small cell lung cancer survival. J Clin Invest 118:2600–2608

    Article  PubMed  CAS  Google Scholar 

  45. Schaefer LK, Ren Z, Fuller GN, Schaefer TS (2002) Constitutive activation of Stat3alpha in brain tumors: localization to tumor endothelial cells and activation by the endothelial tyrosine kinase receptor (VEGFR-2). Oncogene 21:2058–2065

    Article  PubMed  CAS  Google Scholar 

  46. Brantley EC, Benveniste EN (2008) Signal transducer and activator of transcription-3: a molecular hub for signaling pathways in gliomas. Mol Cancer Res 6:675–684

    Article  PubMed  CAS  Google Scholar 

  47. Chen SH, Murphy DA, Lassoued W, Thurston G, Feldman MD, Lee WM (2008) Activated STAT3 is a mediator and biomarker of VEGF endothelial activation. Cancer Biol Ther 7:1994–2003

    Article  PubMed  CAS  Google Scholar 

  48. Wincewicz A, Sulkowska M, Koda M, Leśniewicz T, Kanczuga-Koda L, Sulkowski S (2007) STAT3, HIF-1alpha, EPO and EPOR—signaling proteins in human primary ductal breast cancers. Folia Histochem Cytobiol 45:81–86

    PubMed  CAS  Google Scholar 

  49. Bartoli M, Gu X, Tsai NT, Venema RC, Brooks SE, Marrero MB, Caldwell RB (2000) Vascular endothelial growth factor activates STAT proteins in aortic endothelial cells. J Biol Chem 275:33189–33192

    Article  PubMed  CAS  Google Scholar 

  50. Bartoli M, Platt D, Lemtalsi T, Gu X, Brooks SE, Marrero MB, Caldwell RB (2003) VEGF differentially activates STAT3 in microvascular endothelial cells. FASEB J 17:1562–1564

    PubMed  CAS  Google Scholar 

  51. Saura M, Zaragoza C, Bao C, Herranz B, Rodriguez-Puyol M, Lowenstein CJ (2006) Stat3 mediates interleukin-6 [correction of interleukin-6] inhibition of human endothelial nitric-oxide synthase expression. J Biol Chem 281:30057–30062

    Article  PubMed  CAS  Google Scholar 

  52. Sud N, Kumar S, Wedgwood S, Black SM (2009) Modulation of PKCdelta signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3. Am J Physiol Lung Cell Mol Physiol 296:L519–L526

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (Projects No. 30670834, and No. 30871186), and Research Foundation of the Education Department of Hunan Province, China (project No. 06A060).

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Authors and Affiliations

  1. Center of Biochemistry Research, University of South China, Hengyang, Hunan, 421001, People’s Republic of China

    Limei Yan

  2. Davis Heart & Lung Research Institute and Division of Cardiovascular Medicine, The Ohio State University Medical Center, DHLRI Suite 200, 473 West 12th Ave, Columbus, OH, 43210, USA

    Hong Hao, Terry S. Elton, Zhenguo Liu & Hesheng Ou

  3. College of Pharmacy, Guangxi Medical University, Nanning, 530021, People’s Republic of China

    Hesheng Ou

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  1. Limei Yan
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  2. Hong Hao
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  4. Zhenguo Liu
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Correspondence to Zhenguo Liu or Hesheng Ou.

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Yan, L., Hao, H., Elton, T.S. et al. Intronic microRNA suppresses endothelial nitric oxide synthase expression and endothelial cell proliferation via inhibition of STAT3 signaling. Mol Cell Biochem 357, 9–19 (2011). https://doi.org/10.1007/s11010-011-0870-x

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  • DOI: https://doi.org/10.1007/s11010-011-0870-x

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