Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils

Zhu,Xin‑Hua; Liao,Bing; Xu,Yi; Liu,Ke; Huang,Yun; Huang,Quan‑Long; Liu,Yue‑Hui

doi:10.3892/mmr.2016.6085

Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils

Authors:
- Xin‑Hua Zhu
- Bing Liao
- Yi Xu
- Ke Liu
- Yun Huang
- Quan‑Long Huang
- Yue‑Hui Liu
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Affiliations: Department of Otolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China, Department of Head and Neck Surgery, The Tumor Hospital of Jiangxi Province, Nanchang, Jiangxi 330006, P.R. China
Published online on: December 29, 2016 https://doi.org/10.3892/mmr.2016.6085
Pages: 696-702
Copyright: © Zhu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

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1	Brozek JL, Bousquet J, Baena-Cagnani CE, Bonini S, Canonica GW, Casale TB, van Wijk RG, Ohta K, Zuberbier T and Schünemann HJ: Allergic rhinitis and its impact on asthma (ARIA) guidelines: 2010 revision. J Allergy Clin Immunol. 126:466–476. 2010. View Article : Google Scholar : PubMed/NCBI
2	Zhang Y and Zhang L: Prevalence of allergic rhinitis in China. Allergy Asthma Immunol Res. 6:105–113. 2014. View Article : Google Scholar : PubMed/NCBI
3	Plaut M and Valentine MD: Clinical practice. Allergic rhinitis. N Engl J Med. 353:1934–1944. 2005. View Article : Google Scholar : PubMed/NCBI
4	Weller PF: The immunobiology of eosinophils. N Engl J Med. 324:1110–1118. 1991. View Article : Google Scholar : PubMed/NCBI
5	Stone KD, Prussin C and Metcalfe DD: IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol. 125(2 Suppl 2): S73–S80. 2010. View Article : Google Scholar : PubMed/NCBI
6	Ponath PD, Qin S, Post TW, Wang J, Wu L, Gerard NP, Newman W, Gerard C and Mackay CR: Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J Exp Med. 183:2437–2448. 1996. View Article : Google Scholar : PubMed/NCBI
7	Sallusto F, Mackay CR and Lanzavecchia A: Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science. 277:2005–2007. 1997. View Article : Google Scholar : PubMed/NCBI
8	Fulkerson PC, Fischetti CA, McBride ML, Hassman LM, Hogan SP and Rothenberg ME: A central regulatory role for eosinophils and the eotaxin/CCR3 axis in chronic experimental allergic airway inflammation. Proc Natl Acad Sci USA. 103:16418–16423. 2006. View Article : Google Scholar : PubMed/NCBI
9	Pope SM, Zimmermann N, Stringer KF, Karow ML and Rothenberg ME: The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia. J Immunol. 175:5341–5350. 2005. View Article : Google Scholar : PubMed/NCBI
10	Chuang CC, Su KE, Chen CW, Fan CK, Lin FK, Chen YS and Du WY: Anti-CCR3 monoclonal antibody inhibits eosinophil infiltration in Angiostrongylus cantonensis-infected ICR mice. Acta Trop. 113:209–213. 2010. View Article : Google Scholar : PubMed/NCBI
11	Heath H, Qin S, Rao P, Wu L, LaRosa G, Kassam N, Ponath PD and Mackay CR: Chemokine receptor usage by human eosinophils. The importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J Clin Invest. 99:178–184. 1997. View Article : Google Scholar : PubMed/NCBI
12	Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE and Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391:806–811. 1998. View Article : Google Scholar : PubMed/NCBI
13	DeVincenzo JP: The promise, pitfalls and progress of RNA-interference-based antiviral therapy for respiratory viruses. Antivir Ther. 17:213–225. 2012. View Article : Google Scholar : PubMed/NCBI
14	Karimi MH, Ebadi P, Pourfathollah AA, Moazzeni M, Soheili ZS and Samiee S: Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense and antibody blocking. Hybridoma (Larchmt). 29:473–480. 2010. 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	Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Zhang M, Ihrig MM, McManus MT, et al: A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet. 33:401–406. 2003. View Article : Google Scholar : PubMed/NCBI
17	Dyer KD, Moser JM, Czapiga M, Siegel SJ, Percopo CM and Rosenberg HF: Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J Immunol. 181:4004–4009. 2008. View Article : Google Scholar : PubMed/NCBI
18	Zhu XH, Liao B, Wang XY, Liu K and Liu YH: Construction and identification of mouse eosinophils CCR3gene RNA interference lentiviral vector. Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 48:316–321. 2013.PubMed/NCBI
19	Zhu XH, Liao B, Liu K and Liu YH: Effect of RNA interference therapy on the mice eosinophils CCR3 gene and granule protein in the murine model of allergic rhinitis. Asian Pac J Trop Med. 7:226–230. 2014. View Article : Google Scholar : PubMed/NCBI
20	Tai PC, Sun L and Spry CJ: Effects of IL-5, granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-3 on the survival of human blood eosinophils in vitro. Clin Exp Immunol. 85:312–316. 1991. View Article : Google Scholar : PubMed/NCBI
21	Coffman RL, Seymour BW, Hudak S, Jackson J and Rennick D: Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice. Science. 245:308–310. 1989. View Article : Google Scholar : PubMed/NCBI
22	Cho JY, Miller M, Baek KJ, Han JW, Nayar J, Lee SY, McElwain K, McElwain S, Friedman S and Broide DH: Inhibition of airway remodeling in IL-5-deficient mice. J Clin Invest. 113:551–560. 2004. View Article : Google Scholar : PubMed/NCBI
23	Leckie MJ, ten Brinke A, Khan J, Diamant Z, O'Connor BJ, Walls CM, Mathur AK, Cowley HC, Chung KF, Djukanovic R, et al: Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet. 356:2144–2148. 2000. View Article : Google Scholar : PubMed/NCBI