Original Article

MicroRNAs Expression Induces Apoptosis of Macrophages in Response to Leishmania major (MRHO/IR/75/ER): An In-Vitro and In-Vivo Study


Background: We aimed to investigate the effect of miR-15a mimic and inhibitor of miR-155 expression on apoptosis induction in macrophages infected with Iranian strain of Leishmania major in-vitro and in-vivo.

Methods: RAW 264.7 cells were infected with L. major promastigotes (MRHO/IR/75/ER), and then were treated with miRNAs. For in-vivo experiment, BALB/c mice were inoculated with L. major promastigotes, and then they were treated with miRNAs. For evaluation of miRNA therapeutic effect, in-vitro and in-vivo studies were performed using quantitative Real-time PCR, Flow cytometry, lesion size measurement, and Limiting Dilution Assay (LDA). This study was performed in Shahid Beheshti University of Medical Sciences in 2019.

Results: In-vitro results of flow cytometry showed that using miR-15a mimic, miR-155 inhibitor or both of them increased apoptosis of macrophages. In in-vivo, size of lesion increased during experiment in control groups (P<0.05) while application of both miR-155 inhibitor and miR-15a mimic inhibited the increase in the size of lesions within 6 wk of experiment (P=0.85). LDA results showed that microRNA therapy could significantly decrease parasite load in mimic or inhibitor receiving groups compared to the control group (P<0.05).

Conclusion: miR-155 inhibitor and miR-15a mimic in L. major infected macrophages can induce apoptosis and reduce parasite burden. Therefore, miRNA-based therapy can be proposed as new treatment for cutaneous leishmaniasis.

1. Ghatee MA, Walter RT, Karamian M. The geo-graphical distribution of cutaneous leishmaniasis causative agents in Iran and its neighboring coun-tries, A Review. Front Public Health. 2020; 8: 11.
2. Bailey MS, Lockwood DNJ. Cutaneous leishmania-sis. Clin Dermatol. 2007; 25: 203–211.
3. Holakouie-Naieni K, Mostafavi E, Darvishi Boloo-rani A, et al. Spatial modeling of cutaneous leish-maniasis in Iran from 1983 to 2013. Acta Trop. 2017; 166: 67-73.
4. Gholamrezaei M, Mohebali M, Hanafi-Bojd AA, et al. Ecological niche modeling of main reservoir hosts of zoonotic cutaneous leishmaniasis in Iran. Acta Trop. 2016; 160: 44-52.
5. Duclos S, Desjardins M. Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell Microbiol. 2000; 2(5):365-77.
6. Frank B, Marcu A, Luis de Oliveira Almeida Pe-tersen A, et al. Autophagic digestion of Leishmania major by host macrophages is associated with differ-ential expression of BNIP3, CTSE, and the miR-NAs miR-101c, miR-129, and miR-210. Parasit Vectors. 2015; 8:404.
7. Liu D, Uzonna J E. The early interaction of Leish-mania with macrophages and dendritic cells and its influence on the host immune response. Front Cell Infect Microbiol. 2012; 2:83.
8. Antoine J C, Prina E, Lang Th, et al. The biogenesis and properties of the parasite- ophorous vacuoles that harbor Leishmania in murine macrophages. Trends Microbiol. 1998; 6(10): 392-401.
9. Mohebali M, Kazemirad E, Hajjaran H, et al. Gene expression analysis of antimony resistance in Leish-mania tropica using quantitative real-time PCR fo-cused on genes involved in trypanothione metabo-lism and drug transport. Arch Dermatol Res. 2019; 311(1): 9-17.
10. Mohtasebi S, Mohebali M, Elikaee S, et al. In vitro and in vivo anti-parasitic activity of biogenic antimo-ny sulfide nanoparticles on Leishmania major (MRHO/IR/75/ER). Parasitol Res. 2019; 118(9): 2669-78.
11. Salehi Sangani Gh, Jajarmi V, Khamesipour A, et al. Generation of a CRISPR/Cas9-Based Vector Spe-cific for Gene Manipulation in Leishmania major. Iran J Parasitol. 2019; 14(1): 78-88.
12. Aghaei M, Khanahmad H, Aghaei S, et al. Evolu-tion of transgenic L. infantum expressing mLLO-BAX-SMAC in the infected macrophages apopto-sis in vitro and in vivo. Parasite Immunol. 2020; 42(11):e12726.
13. Vaux DL, Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci U S A. 1996; 93(6): 2239-44.
14. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer. 2002; 2(4): 277-88.
15. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004; 116(2): 281-97.
16. Cai Y, Yu X, Hu S, et al. A Brief Review on the Mechanisms of miRNA Regulation. Genomics Proteomics Bioinformatics. 2009; 7(4): 147-54.
17. Abdullah OA, El Gazzar WB, Salem TI, et al. miR-15a: A Potential Diagnostic Biomarker and a Candi-date for Non-operative Therapeutic Modality for Age-related Cataract. Br J Biomed Sci. 2019; 76(4): 184-189.
18. Zhang H, Li Y, Huang Q, et al. MiR-148a pro-motes apoptosis by targeting Bcl-2 in colorectal cancer. Cell Death Differ. 2011; 18(11): 1702–10.
19. Alizadeh Sh, Kaviani S, Soleimani M, et al. Mir-55 inhibition can reduce cell proliferation and induce apoptosis in Jurkat (Acute T cell Leukemia) cell line. Iran J Ped Hematol Oncol. 2014; 4(4): 141-50.
20. De Santis R, Liepelt A, Mossanen J C, et al. miR-155 targets Caspase-3 mRNA in activated macro-phages. RNA Biol .2016;13(1): 43–58.
21. Kropf P, Kadolsky UD, Rogers M, et al. The Leishmaniasis Model. Methods in Microbiology. 2010; 37: 307–28.
22. Hadighi R, Mohebali M, Boucher P, et al. Unre-sponsiveness to Glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leish-mania tropica parasites. PLoS Med. 2006; 3(5): e162.
23. Laskay T, van Zandbergen G, Solbach W. Neutro-phil granulocytes – Trojan horses for Leishmania ma-jor and other intracellular microbes?. Trends Micro-biol. 2003; 11(5): 210-4.
24. Manzano-Román R, Siles-Lucas M. MicroRNAs in parasitic diseases: potential for diagnosis and target-ing. Mol Biochem Parasitol. 2012; 186(2): 81-6.
25. Liu Q, Tuo W, Gao H, et al. MicroRNAs of para-sites: current status and future perspectives. Parasi-tol Res. 2010; 107(3): 501–07.
26. Sahoo GC, Ansari MY, Dikhit MR, et al. Computa-tional identification of microRNA-like elements in Leishmania major. Microrna. 2014; 2(3): 225-30.
27. Moore KJ, Matlashewsk G. Intracellular infection by Leishmania donovani inhibits macrophage apopto-sis. J Immunol. 1994; 152(6):2930-37.
28. Kaufmann SH, Hengartner MO. Programmed cell death: alive and well in the new millennium. Trends Cell Biol. 2001; 11(12): 526-34.
29. Paris C, Loiseau PM, Bories Ch, et al. Miltefosine Induces apoptosis-like death in Leishmania donovani promastigotes. Antimicrob Agents Chemother. 2004; 48(3): 852-9.
30. Esmaeili J, Mohebali M, Edrissian Gh, et al. Evalua-tion of miltefosine against Leishmania major (MRHO/IR/75/ER): in vitro and in vivo studies. Acta Medica Iranica. 2008; 46(3): 191- 6.
31. Jha TK, Sundar S, Thakur CP, et al. Miltefosine, an oral agent, for the treatment of Indian visceral leishmaniasis. N Engl J Med. 1999; 341(24): 1795-800.
32. Resende TAC, Bernardes VF, Silva JC, et al. Loss of heterozygosity of MIR15A/MIR16-1, negative regulators of the antiapoptotic gene BCL2, is not common in odontogenic keratocysts. Oral Surg Oral Med Oral Pathol Oral Radiol. 2018; 125(4): 313-6.
33. Sicard F, Gayral M, Lulka H, et al. Targeting miR-21for the therapy of pancreatic cancer. Mol Ther. 2013; 21(5): 986-94.
34. Lasjerdi Z, Ghanbarian H, Mohammadi Yeganeh S, et al. The comparative expression profile analysis of apoptosis-related miRNA and its target gene in Leishmania major–infected macrophages. Iran J Para-sitol. 2020;15(3):332-340.
35. Hashemi N, Sharifi MR, Masjedi M, et al. Locked nucleic acid -anti- let-7a induces apoptosis and ne-crosis in macrophages infected with Leishmania ma-jor. Microb Pathog. 2018; 119: 193-99.
IssueVol 15 No 4 (2020) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v15i4.4851
Leishmania major Apoptosis microRNAs In vitro In vivo

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GHOLAMREZAEI M, ROUHANI S, MOHEBALI M, MOHAMMADI-YEGANEH S, HAJI MOLLA HOSEINI M, HAGHIGHI A, LASJERDI Z, HAMIDI F, KAZEM SHARIFI-YAZDI M. MicroRNAs Expression Induces Apoptosis of Macrophages in Response to Leishmania major (MRHO/IR/75/ER): An In-Vitro and In-Vivo Study. Iran J Parasitol. 2020;15(4):475-487.