Expression of miR-15b-5p, miR-21-5p, and SMAD7 in Lung Tissue of Sulfur Mustard-exposed Individuals with Long-term Pulmonary Complications
Sulfur mustard (SM)-exposed individuals develop late pulmonary complications, which are associated with chronic inflammation and fibrotic changes in the lung tissue. MicroRNAs are known to act as important regulators of inflammatory responses, including inflammation and fibrosis-related cytokine signaling. In this study, we investigated the expression miR-15b-5p and miR-21-5p, two regulators of TGF-β signaling, as well as their target molecule, SMAD7, in lung tissues from SM-exposed and control individuals. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue biopsies obtained during surgery from SM-exposed (n=20) or control (n=20) cases. Quality of the extracted RNA was evaluated by an Agilent Bioanalyzer and RNA was quantified using a NanoDrop. MiR-21-5p, miR-15b-5p and SMAD7 expression levels were measured by real-time RT-PCR. miR-21-5p expression levels were significantly decreased (2.7 fold) in the lung tissues from SM-exposed individuals compared with tissues obtained from the control group (p=0.02). There were no significant differences in miR-15b-5p expression levels between the two groups (p=0.94). Interestingly, SMAD7 expression levels were significantly higher (5.8 fold) in SM-exposed individuals’ lung tissues compared with the control group (p=0.045). Our data indicate that exposure to sulfur mustard affects the expression of miR-21-5p as well as its target, SMAD7, in lung tissues many years after exposure. Considering the role of SMAD7 in the regulation of TGF-β signaling, these changes might point to a potential mechanism by which SM-exposure regulates inflammatory/fibrotic alterations in lung tissue.
2. Balali‐Mood M, Hefazi M. Comparison of early and late toxic effects of sulfur mustard in Iranian veterans. Basic Clin Pharmacol Toxicol 2006; 99(4):273-82.
3. Hassan ZM, Ebtekar M, Ghanei M, Taghikhani M, Daloii MRN, Ghazanfari T. Immunobiological consequences of sulfur mustard contamination. Iran J Allergy Asthma Immunol 2006; 5(3):101-8.
4. Ghasemi H, Ghazanfari T, Babaei M, Soroush MR, et al. Long-term ocular complications of sulfur mustard in the civilian victims of Sardasht, Iran. Cutan Ocul Toxicol 2008; 27(4):317-26.
5. Pourfarzam S, Ghazanfari T, Merasizadeh J, Ghanei M, Azimi G, Araghizadeh H, et al. Long-term pulmonary complications in sulfur mustard victims of Sardasht, Iran. Toxin Reviews. 2009;28(1):8-13.
6. Sabourin CL, Rogers JV, Choi YW, Kiser RC, Casillas RP, Babin MC, et al. Time‐and dose‐dependent analysis of gene expression using microarrays in sulfur mustard‐exposed mice. J Biochem Mol Toxicol 2005; 18(6):300-12.
7. Khazdair MR, Boskabady MH, Ghorani V. Respiratory effects of sulfur mustard exposure, similarities and differences with asthma and COPD. Inhal Toxicol 2015; 27(14):731-44.
8. Emad A, Emad Y. Levels of cytokine in bronchoalveolar lavage (BAL) fluid in patients with pulmonary fibrosis due to sulfur mustard gas inhalation. J Interferon Cytokine Res 2007; 27(1):38-43.
9. Ghazanfari T, Kariminia A, Yaraee R, Faghihzadeh S, Ardestani SK, Ebtekar M, et al. Long term impact of sulfur mustard exposure on peripheral blood mononuclear subpopulations—Sardasht-Iran Cohort Study (SICS). Int Immunopharmacol 2013; 17(3):931-5.
10. Shahriary A, Seyedzadeh MH, Ahmadi A, Salimian J. The footprint of TGF-β in airway remodeling of the mustard lung. Inhal Toxicol 2015; 27(14):745-53.
11. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 2003; 425(6958):577-84.
12. Itoh S, Itoh F, Goumans MJ, ten Dijke P. Signaling of transforming growth factor‐β family members through Smad proteins. Eur J Biochem 2000; 267(24):6954-67.
13. O'connell RM, Rao DS, Chaudhuri AA, Baltimore D. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol 2010; 10(2):111-22.
14. Cheng Y, Zhang C. MicroRNA-21 in cardiovascular disease. Journal of cardiovascular translational research. 2010;3(3):251-5.
15. Yan L-X, Huang X-F, Shao Q, Huang M-Y, Deng L, Wu Q-L, et al. MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. Rna. 2008.
16. Lu TX, Munitz A, Rothenberg ME. MicroRNA-21 is up-regulated in allergic airway inflammation and regulates IL-12p35 expression. J Immunol 2009; 182(8):4994-5002.
17. Liu G, Friggeri A, Yang Y, Milosevic J, Ding Q, Thannickal VJ, et al. miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med 2010; 207(8):1589-97.
18. Kumarswamy R, Volkmann I, Thum T. Regulation and function of miRNA-21 in health and disease. RNA Biol 2011; 8(5):706-13.
19. Ozsolak F, Poling LL, Wang Z, Liu H, Liu XS, Roeder RG, et al. Chromatin structure analyses identify miRNA promoters. Genes Dev 2008; 22(22):3172-83.
20. McClelland AD, Herman-Edelstein M, Komers R, Jha JC, Winbanks CE, Hagiwara S, et al. miR-21 promotes renal fibrosis in diabetic nephropathy by targeting PTEN and SMAD7. Clin Sci (Lond) 2015; 129(12):1237-49.
21. Chau BN, Xin C, Hartner J, Ren S, Castano AP, Linn G, et al. MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways. Sci Transl Med 2012; 4(121):121ra18-ra18.
22. Liang H, Zhang C, Ban T, Liu Y, Mei L, Piao X, et al. A novel reciprocal loop between microRNA-21 and TGFβRIII is involved in cardiac fibrosis. Int J Biochem Cell Biol 2012; 44(12):2152-60.
23. Finnerty JR, Wang W-X, Hébert SS, Wilfred BR, Mao G, Nelson PT. The miR-15/107 group of microRNA genes: evolutionary biology, cellular functions, and roles in human diseases. J Mol Biol 2010; 402(3):491-509.
24. Ezzie ME, Crawford M, Cho J-H, Orellana R, Zhang S, Gelinas R, et al. Gene expression networks in COPD: microRNA and mRNA regulation. Thorax 2012; 67(2):122-31.
25. Zandvoort A, Postma DS, Jonker MR, Noordhoek JA, Vos JT, Timens W. Smad gene expression in pulmonary fibroblasts: indications for defective ECM repair in COPD. Respir Res 2008; 9(1):83.
26. Adelipour M, Fooladi AAI, Yazdani S, Vahedi E, Ghanei M, Nourani MR. Smad molecules expression pattern in human bronchial airway induced by sulfur mustard. Iran J Allergy Asthma Immunol 2011; 10(3):147-54.
27. Zarin AA, Behmanesh M, Tavallaei M, Shohrati M, Ghanei M. Overexpression of transforming growth factor (TGF)-β1 and TGF-β3 genes in lung of toxic-inhaled patients. Exp Lung Res 2010; 36(5):284-91.
28. Valizadeh M, Mirzaei B, Tavallaei M, Noorani MR, Amiri M, Soroush MR, et al. Down-regulation of TGF-b1, TGF-b receptor 2, and TGF-b-associated microRNAs, miR-20a and miR-21, in skin lesions of sulfur mustard-exposed Iranian war veterans. J Recept Signal Transduct Res 2015; 35(6):634-9.
29. Seike M, Goto A, Okano T, Bowman ED, Schetter AJ, Horikawa I, et al. MiR-21 is an EGFR-regulated anti-apoptotic factor in lung cancer in never-smokers. Proc Natl Acad Sci U S A 2009; 106(29):12085-90.
30. Mosayebzadeh M, Ghazanfari T, Delshad A, Akbari H. Evaluation of apoptosis in the lung tissue of sulfur mustard-exposed individuals. Iran J Allergy Asthma Immunol 2016; 15(4):283-8.
31. Sheedy FJ. Turning 21: induction of miR-21 as a key switch in the inflammatory response. Front Immunol 2015; 6:19.
32. Ma X, Becker Buscaglia LE, Barker JR, Li Y. MicroRNAs in NF-κB signaling. J Mol Cell Biol 2011; 3(3):159-66.
33. Molina-Pinelo S, Pastor MD, Suarez R, Romero-Romero B, De la Peña MG, Salinas A, et al. MicroRNA clusters: dysregulation in lung adenocarcinoma and COPD. Eur Respir J 2014; 43(6):1740-9.
34. Ezzie ME, Crawford M, Cho J-H, Orellana R, Zhang S, Gelinas R, et al. Gene expression networks in COPD: microRNA and mRNA regulation. Thorax 2012; 67(2):122-31.