shRNAs targeting high‑mobility group box-1 inhibit E-selectin expression via homeobox A9 in human umbilical vein endothelial cells

Zhang,Xiao-Juan; Luan,Zheng-Gang; Ma,Xiao-Chun

doi:10.3892/mmr.2013.1314

shRNAs targeting high‑mobility group box-1 inhibit E-selectin expression via homeobox A9 in human umbilical vein endothelial cells

Authors:
- Xiao-Juan Zhang
- Zheng-Gang Luan
- Xiao-Chun Ma
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Affiliations: Intensive Care Unit, First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China
Published online on: February 7, 2013 https://doi.org/10.3892/mmr.2013.1314
Pages: 1251-1256

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Abstract

The nucleus of the endothelial cell contains large amounts of high‑mobility group box protein 1 (HMGB1), a cytokine mediator of inflammation, and the endothelium may be a crucial source of HMGB1 during the inflammatory response. Therefore, the downregulation of HMGB1 expression by RNA interference (RNAi) may decrease inflammatory activity. The aim of this study was to investigate the possible mechanism of action and the effect of HMGB1 on homeobox A9 (HOXA9) and E-selectin expression. Recombinant human full-length HMGB1 was cloned by PCR amplification from human umbilical vein endothelial cells (HUVECs) and then subcloned into a pcDNA‑3.1-myc-his-B vector. Specific short hairpin RNAs (shRNAs) for the HMGB1 target sequence and a scrambled sequence were designed, synthesized and cloned into a pRNA‑U6.1/Neo vector. Specific small interfering RNAs (siRNAs) for the HOXA9 target sequence were commercially prepared and the expression levels of HMGB1, HOXA9, intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and E-selectin were detected using real-time PCR and western blot analysis. The expression of the full-length HMGB1 gene and protein was verified in HUVECs. The shRNA for HMGB1 and siRNA for HOXA9 successfully decreased the expression levels of HMGB1 and HOXA9, respectively. ICAM1, VCAM1 and E-selectin were downregulated through HMGB1 interference in HUVECs, and HMGB1 shRNA decreased E-selectin expression by HOXA9. These results demonstrated the potential use of specific siRNA targeting HMGB1 expression for the development of novel therapeutic agents for inflammatory disorders.

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1	Skoko D, Wong B, Johnson RC and Marko JF: Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes. Biochemistry. 43:13867–13874. 2004. View Article : Google Scholar : PubMed/NCBI
2	Pallier C, Scaffidi P, Chopineau-Proust S, et al: Association of chromatin proteins high mobility group box (HMGB) 1 and HMGB2 with mitotic chromosomes. Mol Biol Cell. 14:3414–3426. 2003. View Article : Google Scholar : PubMed/NCBI
3	Yuan F, Gu L, Guo S, Wang C and Li GM: Evidence for involvement of HMGB1 protein in human DNA mismatch repair. J Biol Chem. 279:20935–20940. 2004. View Article : Google Scholar : PubMed/NCBI
4	Andersson U, Wang H, Palmblad K, et al: High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med. 192:565–570. 2000. View Article : Google Scholar : PubMed/NCBI
5	Fiuza C, Bustin M, Talwar S, et al: Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood. 101:2652–2660. 2003. View Article : Google Scholar : PubMed/NCBI
6	Wang H, Bloom O, Zhang M, et al: HMG-1 as a late mediator of endotoxin lethality in mice. Science. 285:248–251. 1999. View Article : Google Scholar : PubMed/NCBI
7	Wang H, Yang H and Tracey KJ: Extracellular role of HMGB1 in inflammation and sepsis. J Intern Med. 255:320–331. 2004. View Article : Google Scholar : PubMed/NCBI
8	Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J and Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med. 29:1303–1310. 2001. View Article : Google Scholar : PubMed/NCBI
9	Lehr HA, Bittinger F and Kirkpatrick CJ: Microcirculatory dysfunction in sepsis: a pathogenetic basis for therapy? J Pathol. 190:373–386. 2000. View Article : Google Scholar : PubMed/NCBI
10	Bae JS and Rezaie AR: Activated protein C inhibits high mobility group box 1 signaling in endothelial cells. Blood. 118:3952–3959. 2011. View Article : Google Scholar : PubMed/NCBI
11	Mullins GE, Sunden-Cullberg J, Johansson AS, et al: Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1. Scand J Immunol. 60:566–573. 2004. View Article : Google Scholar : PubMed/NCBI
12	Yang H, Ochani M, Li J, et al: Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci USA. 101:296–301. 2004. View Article : Google Scholar : PubMed/NCBI
13	Qin S, Wang H, Yuan R, et al: Role of HMGB1 in apoptosis-mediated sepsis lethality. J Exp Med. 203:1637–1642. 2006. View Article : Google Scholar : PubMed/NCBI
14	Paul CP, Good PD, Winer I and Engelke DR: Effective expression of small interfering RNA in human cells. Nat Biotechnol. 20:505–508. 2002. View Article : Google Scholar : PubMed/NCBI
15	Paddison PJ, Caudy AA, Bernstein E, Hannon GJ and Conklin DS: Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16:948–958. 2002. View Article : Google Scholar : PubMed/NCBI
16	Kuniyasu H, Chihara Y and Kondo H: Differential effects between amphoterin and advanced glycation end products on colon cancer cells. Int J Cancer. 104:722–727. 2003. View Article : Google Scholar : PubMed/NCBI
17	Shapiro NI, Yano K, Sorasaki M, Fischer C, Shih SC and Aird WC: Skin biopsies demonstrate site-specific endothelial activation in mouse models of sepsis. J Vasc Res. 46:495–502. 2009. View Article : Google Scholar : PubMed/NCBI
18	Nooteboom A, van der Linden CJ and Hendriks T: Modulation of adhesion molecule expression on endothelial cells after induction by lipopolysaccharide-stimulated whole blood. Scand J Immunol. 59:440–448. 2004. View Article : Google Scholar : PubMed/NCBI
19	Sessler CN, Windsor AC, Schwartz M, et al: Circulating ICAM-1 is increased in septic shock. Am J Respir Crit Care Med. 151:1420–1427. 1995. View Article : Google Scholar : PubMed/NCBI
20	Bandyopadhyay S, Ashraf MZ, Daher P, Howe PH and DiCorleto PE: HOXA9 participates in the transcriptional activation of E-selectin in endothelial cells. Mol Cell Biol. 27:4207–4216. 2007. View Article : Google Scholar : PubMed/NCBI
21	Trivedi CM, Patel RC and Patel CV: Differential regulation of HOXA9 expression by nuclear factor kappa B (NF-kappaB) and HOXA9. Gene. 408:187–195. 2008. View Article : Google Scholar : PubMed/NCBI
22	Weissberg PL and Bennett MR: Atherosclerosis - an inflammatory disease. N Engl J Med. 340:1928–1929. 1999. View Article : Google Scholar : PubMed/NCBI
23	Chen JW, Chen YH, Lin FY, Chen YL and Lin SJ: Ginkgo biloba extract inhibits tumor necrosis factor-alpha-induced reactive oxygen species generation, transcription factor activation, and cell adhesion molecule expression in human aortic endothelial cells. Arterioscler Thromb Vasc Biol. 23:1559–1566. 2003. View Article : Google Scholar : PubMed/NCBI
24	Patel CV, Sharangpani R, Bandyopadhyay S and DiCorleto PE: Endothelial cells express a novel, tumor necrosis factor-alpha-regulated variant of HOXA9. J Biol Chem. 274:1415–1422. 1999. View Article : Google Scholar : PubMed/NCBI
25	Ross R: The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 362:801–809. 1993. View Article : Google Scholar : PubMed/NCBI
26	Tracey KJ, Beutler B, Lowry SF, et al: Shock and tissue injury induced by recombinant human cachectin. Science. 234:470–474. 1986. View Article : Google Scholar : PubMed/NCBI
27	Hesse DG, Tracey KJ, Fong Y, et al: Cytokine appearance in human endotoxemia and primate bacteremia. Surg Gynecol Obstet. 166:147–153. 1988.PubMed/NCBI
28	Beutler BA, Milsark IW and Cerami A: Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J Immunol. 135:3972–3977. 1985.PubMed/NCBI
29	Fink MP: Another negative clinical trial of a new agent for the treatment of sepsis: rethinking the process of developing adjuvant treatments for serious infections. Crit Care Med. 23:989–991. 1995. View Article : Google Scholar : PubMed/NCBI
30	Fisher CJ Jr, Dhainaut JF, Opal SM, et al: Phase III rhIL-1ra Sepsis Syndrome Study Group: Recombinant human interleukin 1 receptor antagonist in the treatment of patients with sepsis syndrome. Results from a randomized, double-blind, placebo-controlled trial. JAMA. 271:1836–1843. 1994. View Article : Google Scholar
31	Eskandari MK, Bolgos G, Miller C, Nguyen DT, DeForge LE and Remick DG: Anti-tumor necrosis factor antibody therapy fails to prevent lethality after cecal ligation and puncture or endotoxemia. J Immunol. 148:2724–2730. 1992.
32	Remick D, Manohar P, Bolgos G, Rodriguez J, Moldawer L and Wollenberg G: Blockade of tumor necrosis factor reduces lipopolysaccharide lethality, but not the lethality of cecal ligation and puncture. Shock. 4:89–95. 1995. View Article : Google Scholar : PubMed/NCBI
33	Luan ZG, Zhang H, Yang PT, Ma XC, Zhang C and Guo RX: HMGB1 activates nuclear factor-κB signaling by RAGE and increases the production of TNF-α in human umbilical vein endothelial cells. Immunobiology. 215:956–962. 2010.
34	Unoshima M: Therapeutic effect of anti-HMGB1 antibody and anti-RAGE antibody on SIRS/sepsis. Nihon Rinsho. 62:2323–2329. 2004.(In Japanese).
35	Parrish W and Ulloa L: High-mobility group box-1 isoforms as potential therapeutic targets in sepsis. Methods Mol Biol. 361:145–162. 2007.PubMed/NCBI
36	Eriksson M: Should high mobility group box-1 protein (HMGB1) be measured in patients with severe sepsis and septic shock? If so, when, where, and how? Crit Care Med. 33:682–683. 2005. View Article : Google Scholar : PubMed/NCBI
37	Ulloa L, Ochani M, Yang H, et al: Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci USA. 99:12351–12356. 2002. View Article : Google Scholar : PubMed/NCBI
38	Guo RF and Ward PA: Role of C5a in inflammatory responses. Annu Rev Immunol. 23:821–852. 2005. View Article : Google Scholar : PubMed/NCBI
39	Mantell LL, Parrish WR and Ulloa L: HMGB-1 as a therapeutic target for infectious and inflammatory disorders. Shock. 25:4–11. 2006. View Article : Google Scholar : PubMed/NCBI
40	Ulloa L and Messmer D: High-mobility group box 1 (HMGB1) protein: friend and foe. Cytokine Growth Factor Rev. 17:189–201. 2006. View Article : Google Scholar : PubMed/NCBI
41	Ferrari S, Finelli P, Rocchi M and Bianchi ME: The active gene that encodes human high mobility group 1 protein (HMG1) contains introns and maps to chromosome 13. Genomics. 35:367–371. 1996. View Article : Google Scholar : PubMed/NCBI
42	Kornblit B, Munthe-Fog L, Petersen SL, Madsen HO, Vindelov L and Garred P: The genetic variation of the human HMGB1 gene. Tissue Antigens. 70:151–156. 2007. View Article : Google Scholar : PubMed/NCBI
43	Bustin M and Reeves R: High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. Prog Nucleic Acid Res Mol Biol. 54:35–100. 1996. View Article : Google Scholar : PubMed/NCBI
44	Ferrari S, Ronfani L, Calogero S and Bianchi ME: The mouse gene coding for high mobility group 1 protein (HMG1). J Biol Chem. 269:28803–28808. 1994.PubMed/NCBI
45	Hardman CH, Broadhurst RW, Raine AR, Grasser KD, Thomas JO and Laue ED: Structure of the A-domain of HMG1 and its interaction with DNA as studied by heteronuclear three- and four-dimensional NMR spectroscopy. Biochemistry. 34:16596–16607. 1995. View Article : Google Scholar : PubMed/NCBI
46	Kuhns DB, Alvord WG and Gallin JI: Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans. J Infect Dis. 171:145–152. 1995. View Article : Google Scholar
47	Boldt J, Muller M, Kuhn D, Linke LC and Hempelmann G: Circulating adhesion molecules in the critically ill: a comparison between trauma and sepsis patients. Intensive Care Med. 22:122–128. 1996. View Article : Google Scholar : PubMed/NCBI
48	Kayal S, Jais JP, Aguini N, Chaudiere J and Labrousse J: Elevated circulating E-selectin, intercellular adhesion molecule 1, and von Willebrand factor in patients with severe infection. Am J Respir Crit Care Med. 157:776–784. 1998. View Article : Google Scholar : PubMed/NCBI
49	Harlan JM and Winn RK: Leukocyte-endothelial interactions: clinical trials of anti-adhesion therapy. Crit Care Med. 30:S214–S219. 2002. View Article : Google Scholar : PubMed/NCBI
50	Treutiger CJ, Mullins GE, Johansson AS, et al: High mobility group 1 B-box mediates activation of human endothelium. J Intern Med. 254:375–385. 2003. View Article : Google Scholar : PubMed/NCBI
51	Zappavigna V, Falciola L, Helmer-Citterich M, Mavilio F and Bianchi ME: HMG1 interacts with HOX proteins and enhances their DNA binding and transcriptional activation. EMBO J. 15:4981–4991. 1996.PubMed/NCBI
52	Mark M, Rijli FM and Chambon P: Homeobox genes in embryogenesis and pathogenesis. Pediatr Res. 42:421–429. 1997. View Article : Google Scholar : PubMed/NCBI
53	Trivedi CM, Patel RC and Patel CV: Homeobox gene HOXA9 inhibits nuclear factor-kappa B dependent activation of endothelium. Atherosclerosis. 195:50–60. 2007. View Article : Google Scholar : PubMed/NCBI