Original Article

Differential Expression of miR-21-5p, miR-20a-5p, TGF-β1, and TGF-β Receptor 2 in Skin, Serum, and Lung Samples Exposed to Sulfur Mustard


Sulfur mustard (SM) or mustard gas is a blister chemical agent that causes pulmonary damage by triggering inflammation and oxidative injury. Alterations in microRNA (miR) transcript levels are found in pulmonary diseases and even inflammation. Therefore, we evaluated the expression levels of miR-20a-5p, miR-21-5p, and two target transcripts (transforming growth factor-beta [TGF-β1] and TGF-β receptor 2 [TGFR2]) in lung, serum, and skin samples from patients exposed to SM.
Total RNA was extracted from lung, serum, and skin samples of patients with moderate (n=10) and high (n=10) SM exposure, as well as 10 healthy subjects. Following the synthesis of complementary deoxyribonucleic acid using real-time polymerase chain reaction, we determined the expression levels of miR-20a-5p, miR-21-5p, TGF-β1, and TGFR2 transcripts. Furthermore, we evaluated the sensitivity and specificity of the chosen miRs by employing receiver operating characteristic (ROC) curves and calculating the area under the ROC curve.
The results showed that miR-20a-5p and miR-21-5p expressions in the groups with moderate and high SM exposure were significantly lower than the normal controls. The expression analysis demonstrated that TGFR2 was significantly less expressed in skin samples exposed to SM in both groups of patients compared with healthy controls. Furthermore, the TGF-β1 expression in the skin samples of the group with moderate SM exposure was lower than that of the normal control group.
Our findings suggest that miR-20a-5p, miR-21-5p, TGF-β1, and TGFR2 expressions could be used as potential biomarkers for discriminating SM-exposed patients from healthy individuals.

1. Selcuklu SD, Donoghue MT, Spillane C. miR-21 as a key regulator of oncogenic processes. Biochem Soc Trans. 2009; 37(4):918-25.
2. Kehe K, Balszuweit F, Emmler J, Kreppel H, Jochum M, Thiermann H. Sulfur mustard research—strategies for the development of improved medical therapy. Eplasty. 2008; 8: e32.
3. Ghasemi H, Owlia P, Jalali-Nadoushan MR, Pourfarzam S, Azimi G, Yarmohammadi ME, et al. A clinicopathological approach to sulfur mustard-induced organ complications: a major review. Cutan Ocul Toxicol. 2013; 32(4):304-24.
4. Graham JS, Schoneboom BA. Historical perspective on effects and treatment of sulfur mustard injuries. Chem Biol Interact. 2013; 206(3):512-22.
5. Gao X, Ray R, Xiao Y, Barker PE, Ray P. Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells. BMC Cell Biol. 2007;8:17.
6. Sadeghi S, Tapak M, Ghazanfari T, Mosaffa N. A review of Sulfur Mustard-induced pulmonary immunopathology: An Alveolar Macrophage Approach. Toxicol Lett. 2020;333:115-29.
7. 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.
8. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 2014;15:509-24.
9. Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol. 2007;23:175-205.
10. Butz H, Rácz K, Hunyady L, Patócs A. Crosstalk between TGF-β signaling and the microRNA machinery. Trends Pharmacol Sci. 2012;33(7):382-93.
11. Xiao Z, Chen S, Feng S, Li Y, Zou J, Ling H, et al. Function and mechanisms of microRNA 20a in colorectal cancer. Exp Ther Med. 2020;19(3):1605-16.
12. Jiang Y, Chang H, Chen G. Effects of microRNA 20a on the proliferation, migration and apoptosis of multiple myeloma via the PTEN/PI3K/AKT signaling pathway. Oncol Lett. 2018;15(6):10001-7.
13. Yang X, Guo Y, Du Y, Yang J, Li S, Liu S, et al. Serum microRNA-21 as a diagnostic marker for lung carcinoma: a systematic review and meta-analysis. PloS One. 2014;9(5):e97460.
14. Sheedy FJ. Turning 21: induction of miR-21 as a key switch in the inflammatory response. Front Immunol. 2015;6:19.
15. Krichevsky AM, Gabriely G. miR-21: a small multi-faceted RNA. J Cell Mol Med. 2009;13:39-53.
16. Weinberger B, Laskin JD, Sunil VR, Sinko PJ, Heck DE, Laskin DL. Sulfur mustard-induced pulmonary injury: therapeutic approaches to mitigating toxicity. Pulm Pharmacol Ther. 2011;24(1):92-9.
17. Suzuki HI. MicroRNA control of TGF-β signaling. Int J Mol Sci. 2018;19(7):1901.
18. Rana T, Ahmad A, Zafar I, Mariappan N, Chandrashekar DS, Hamid T, et al. MicroRNA-mediated inflammation and coagulation effects in rats exposed to an inhaled analog of sulfur mustard. Ann NY Acad Sci. 2020;1479(1): 148-58.
19. 19Menacher G, Steinritz D, Schmidt A, Popp T, Worek F, Gudermann T, et al. Effects of anti-inflammatory compounds on sulfur mustard injured cells: Recommendations and caveats suggested by in vitro cell culture models. Toxicol Lett. 2018;293:91-7.
20. Malaviya R, Sunil VR, Cervelli J, Anderson DR, Holmes WW, Conti ML, et al. Inflammatory effects of inhaled sulfur mustard in rat lung. Toxicol Appl Pharmacol. 2010; 248(2):89-99.
21. Jahromy MG, Mohsenian MS, Rezagholizamenjany M, Ansari I, Mirzadeh SM, Nejad-Moghaddam A. Effects of mustard gas on veterans: a review. Ann Mil Health Sci Res. 2017;15(2):e62051.
22. Alvanegh AG, Edalat H, Fallah P, Tavallaei M. Decreased expression of miR-20a and miR-92a in the serum from sulfur mustard-exposed patients during the chronic phase of resulting illness. Inhal Toxicol. 2015;27(13):682-28.
23. Wei J, Gao W, Zhu CJ, Liu YQ, Mei Z, Cheng T, et al. Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non–small cell lung cancer. Chin J Cancer. 2011;30(6):407-14.
24. Davis BN, Hilyard AC, Lagna G, Hata A. SMAD proteins control DROSHA-mediated microRNA maturation. Nature. 2008;454(7200):56-61.
25. Yuan J, Li P, Pan H, Li Y, Xu Q, Xu T, et al. miR-542-5p attenuates fibroblast activation by targeting integrin α6 in silica-induced pulmonary fibrosis. Int J Mol Sci. 2018;19(12):3717.
26. Arabipour I, Amani J, Mirhosseini SA, Salimian J. The study of genes and signal transduction pathways involved in mustard lung injury: A gene therapy approach. Gene. 2019;714:143968.
27. Salimi S, Noorbakhsh F, Faghihzadeh S, Ghaffarpour S, Ghazanfari T. Expression of miR-15b-5p, miR-21-5p, and SMAD7 in lung tissue of sulfur mustard-exposed individuals with long-term pulmonary complications. Iran J Allergy Asthma Immunol. 2019;18(3):332-9.
28. Adelipour M, Imani Fooladi AI, Yazdani S, Vahedi E, Ghanei M, Nourani MR. Smad molecules expression pattern in human bronchial airway induced by sulfur mustard. Iran J Allergy Asthma Imunol. 2011;10(3):147-54.
29. Ma X, Buscaglia LE, Barker JR, Li Y. MicroRNAsin NF-B signaling. J Mol Cell Biol. 2011;3(3):159-66.
30. Kunita A, Morita S, Irisa TU, Goto A, Niki T, Takai D, et al. MicroRNA-21 in cancer-associated fibroblasts supports lung adenocarcinoma progression. Sci Rep. 2018;8(1):8838.
31. Sałaga M, Fichna J, Socała K, Nieoczym D, Pieróg M, Zielińska M, et al. Neuropharmacological characterization of the oneirogenic Mexican plant Calea zacatechichi aqueous extract in mice. Metab Brain Dis. 2016;31:631-41.
32. Everett JAC. The 12 item social and economic conservatism scale (SECS). PloS One. 2013;8(12): e82131.
33. Wei L, Ran F. MicroRNA-20a promotes proliferation and invasion by directly targeting early growth response 2 in non small cell lung carcinoma. Oncol Lett. 2018;15(1):271-7.
34. Liu Z, Yu H, Guo Q. MicroRNA 20a promotes inflammation via the nuclear factor κB signaling pathway in pediatric pneumonia. Mol Med Rep. 2018;17(1):612-7.
35. Li XF, Shen WW, Sun YY, Li WX, Sun ZH, Liu YH, et al. MicroRNA-20a negatively regulates expression of NLRP3-inflammasome by targeting TXNIP in adjuvant-induced arthritis fibroblast-like synoviocytes. Joint Bone Spine. 2016;83(6):695-700.
36. Liang B, Wang X, Song X, Bai R, Yang H, Yang Z, et al. MicroRNA-20a/b regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1. Biochim Biophys Acta Mol Cell Biol Lipids. 2017;1862(9):929-38.
37. Zhu D, Pan C, Li L, Bian Z, Lv Z, Shi L, et al. MicroRNA-17/20a/106a modulate macrophage inflammatory responses through targeting signal-regulatory protein α. J Allergy Clin Immunol. 2013;132(2):426-36.
IssueVol 22 No 4 (2023) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijaai.v22i4.13609
Gene expression MicroRNAs Mustard gas Transforming growth factor beta1

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Valizadeh M, Irani S, Tavallaei M, Soleimani M. Differential Expression of miR-21-5p, miR-20a-5p, TGF-β1, and TGF-β Receptor 2 in Skin, Serum, and Lung Samples Exposed to Sulfur Mustard. Iran J Allergy Asthma Immunol. 2023;22(4):366-378.