Molecular and Cellular Biochemistry

, Volume 390, Issue 1–2, pp 115–121 | Cite as

Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro



The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNL-HSP27-EGFP-2 (all P < 0.01). To conclude, these data indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases.


Heat shock protein 27(HSP27) RNA interference (RNAi) Vascular smooth muscle cells (VSMCs) Cytoskeleton Migration 



This work was supported by Grants from the Clinical Key Program of the Fujian Medical University (XK201107) and the Fujian Natural Science Foundation (2011J01160).


  1. 1.
    Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316PubMedGoogle Scholar
  2. 2.
    Owens GK, Wise G (1997) Regulation of differentiation/maturation in vascular smooth muscle cells by hormones and growth factors. Agents Actions Suppl48:3–24Google Scholar
  3. 3.
    Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84:767–780PubMedCrossRefGoogle Scholar
  4. 4.
    Davis-Dusenbery BN, Wu C, Hata A (2011) Micromanaging vascular smooth muscle cell differentiation and phenotypic modulation. Arterioscler Thromb Vasc Biol 31:2370–2377PubMedCrossRefGoogle Scholar
  5. 5.
    Rensen SS, Doevendans PA, van Eys GJ (2007) Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J 15:100–108PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Tallquist M, Kazlauskas A (2004) PDGF signaling in cells and mice. Cytokine Growth Factor Rev 15:205–213PubMedCrossRefGoogle Scholar
  7. 7.
    Ciocca DR, Oesterreich S, Chamness GC, McGuire WL, Fuqua SA (1993) Biological and clinical implications of heat shock protein 27,000 (HSP27): a review. J Natl Cancer Inst 85(19):1558–1570PubMedCrossRefGoogle Scholar
  8. 8.
    Benndorf R, Hayess K, Ryazantsev S, Wieske M, Behlke J, Lutsch G (1994) Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. J Biol Chem 269(32):20780–20784PubMedGoogle Scholar
  9. 9.
    Miron T, Vancompernolle K, Vandekerckhove J, Wilchek M, Geiger BA (1991) 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. J Cell Biol 114:255–261PubMedCrossRefGoogle Scholar
  10. 10.
    Bitar KN (2002) HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction. Am J Physiol Gastrointest Liver Physiol 282(5):G894–G903PubMedGoogle Scholar
  11. 11.
    Chen HF, Xie LD, Xu CS (2009) Role of heat shock protein 27 phosphorylation in migration of vascular smooth muscle cells. Mol Cell Biochem 327(1–2):1–6PubMedCrossRefGoogle Scholar
  12. 12.
    Chen HF, Xie LD, Xu CS (2010) The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells. Mol Cell Biochem 333:49–56PubMedCrossRefGoogle Scholar
  13. 13.
    Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59:75–86PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498PubMedCrossRefGoogle Scholar
  15. 15.
    Sohail M, Doran G, Riedemann J, Macaulay V (2003) Southern EM.A simple and cost-effective method for producing small interfering RNAs with high efficacy. Nucleic Acids Res 31(7):e38PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Meister G, Tuschl T (2004) Mechanisms of gene silencing by double stranded RNA. Nature 431:343–349PubMedCrossRefGoogle Scholar
  17. 17.
    McManus MT, Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3:737–747PubMedCrossRefGoogle Scholar
  18. 18.
    Huang J, Xie LD, Xu CS, Wang HJ (2009) Construction and identification of a recombinant lentiviral vector harbouring RNAi targeting rat HSP27 gene. Chin Pharmacol Bull 25(4):488–492Google Scholar
  19. 19.
    Huang J, Xie LD (2008) Expression of human apM1 lentiviral vector in 293 T cell and it’s significance. Chin Pharmacol Bull 24(7):928–932Google Scholar
  20. 20.
    Yu HZ, Xie L, Zhu P, Xu C, Wang H-J (2012) Human tissue kallikrein 1 gene delivery inhibits PDGF-BB-induced vascular smooth muscle cells proliferation and upregulates the expressions of p27Kip1 and p2lCip1. Mol Cell Biochem 360:363–371PubMedCrossRefGoogle Scholar
  21. 21.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Ross R (1999) Atherosclerosis is an inflammatory disease. Am Heart J 138:S419–S420PubMedCrossRefGoogle Scholar
  23. 23.
    Braun-Dullaeus RC, Mann MJ, Dzau VJ (1998) Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation 98(1):82–89PubMedCrossRefGoogle Scholar
  24. 24.
    Sriram V, Patterson C (2001) Cell cycle in vasculoproliferative diseases: potential interventions and routes of delivery. Circulation 103(19):2414–2419PubMedCrossRefGoogle Scholar
  25. 25.
    Rousseau S, Houle F, Landry J, Huot J (1997) p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15:2169–2177PubMedCrossRefGoogle Scholar
  26. 26.
    Park HK, Park EC, Bae SW et al (2006) Expression of heat shock protein 27 in human atherosclerotic plaques and increased plasma level of heat shock protein 27 in patients with acute coronary syndrome. Circulation 114:886–893PubMedCrossRefGoogle Scholar
  27. 27.
    Tallot P, Grongnet JF, David JC (2003) Dual perinatal and developmental expression of the small heat shock proteins [FC12] aB-crystallin and HSP27 in different tissues of the developing piglet. Biol Neonate 83:281–288PubMedCrossRefGoogle Scholar
  28. 28.
    Tartakover-Matalon S, Cherepnin N, Kuchuk M et al (2007) Impaired migration of trophoblast cells caused by simvastatin is associated with decreased membrane IGF-I receptor, MMP-2 activity and HSP27 expression. Hum Reprod 22(4):1161–1167PubMedCrossRefGoogle Scholar
  29. 29.
    Lee CK, Lee HM, Kim HJ et al (2007) Syk contributes to PDGFBB-mediated migration of rat aortic smooth muscle cells via MAPK pathways. Cardiovasc Res 74(1):159–168PubMedCrossRefGoogle Scholar
  30. 30.
    Purushothaman KR, Meerarani P, Moreno PR (2007) Inflammation and neovascularization in diabetic atherosclerosis. Indian J Exp Biol 45(1):93–102PubMedGoogle Scholar
  31. 31.
    Somara S, Bitar KN (2004) Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle. Am J Physiol Cell Physiol 286:C1290–C1301PubMedCrossRefGoogle Scholar
  32. 32.
    Chen Y, Currie RW (2006) Small interfering RNA knocks down heat shock factor-1(HSF-1) and exacerbates pro-inflammatory activation of NF-kB and AP-1 in vascular smooth muscle cells. Cardiovasc Res 69:66–75PubMedCrossRefGoogle Scholar
  33. 33.
    Huot J, Houle F, Spitz DR, Landry J (1996) HSP27 phosphorylation mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 56:273–279PubMedGoogle Scholar
  34. 34.
    Doshi BM, Hightower LE, Lee J (2009) The role of HSP27 and actin in the regulation of movement in human cancer cells responding to heat shock. Cell Stress Chaperones 14:445–457PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Hedges JC, Dechert MA, Yamboliev IA et al (1999) A role for p38MAPK/HSP27 pathway in smooth muscle cell migration. Biol Chem 274(34):24211–24219CrossRefGoogle Scholar
  36. 36.
    Rosner D, McCarthy N, Bennett M (2005) Rapamycin inhibits human in stent restenosis vascular smooth muscle cells independently of pRB phosphorylation and p53. Cardiovasc Res 66(3):601–610PubMedCrossRefGoogle Scholar
  37. 37.
    Sun J, Zheng J, Ling KH et al (2012) Preventing intimal thickening of vein grafts in vein artery bypass using STAT-3 siRNA. J Transl Med 10:2PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Fujian Hypertension Research InstituteFirst Affiliated Hospital of Fujian Medical UniversityFuzhou China
  2. 2.Department of Cardiology of the ZhengzhouCentral Hospital Affiliated to Zhengzhou UniversityZhengzhouChina

Personalised recommendations