Iranian Journal of Allergy, Asthma and Immunology 2017. 16(3):235-244.

Effect of MicroRNA-21 Transfection on In-vitro Differentiation of Human Naive CD4+ T Cells to Regulatory T Cells
Haideh Namdari, Mojgan Ghayedi, Jamshid Hadjati, Farhad Rezaei, Kurosh Kalantar, Parisa Rahimzadeh, Eisa Salehi


Regulatory T cells (Tregs) are important components of the immune system that modulate responses of other cells. These cells are involved in peripheral tolerance mechanisms, so defect in development and function of these cells can result in autoimmune disease. Increasing evidence supports the role of microRNAs-21 (miR-21) in the regulation of forkhead box P3 (Foxp3) expression in Tregs. We aimed to determine whether miR-21 transfection to naive CD4+ T cells can be useful in generation of iTregs in-vitro. We investigated in-vitro differentiation of miR-21-transfected naive CD4+ T cells to iTregs and compared these iTregs to cytokine-differentiated iTregs and control group. We showed that expression of Foxp3, transforming growth factor beta (TGF-β), and interleukin-10 (IL-10) are increased in iTregs generated after miR-21 transfection in comparison with cytokine-differentiated iTregs and control group. Our findings demonstrate that miR-21 has positive role in in-vitro generation of induced regulatory T-cells (iTregs).



Foxp3; Mir-21, In-vitro differentiation; Naive CD4-positive T-lymphocytes; Regulatory T lymphocyte

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1.       Yang, W.Y., Y. Shao, J. Lopez-Pastrana, J. Mai, H. Wang, and X.-f. Yang, Pathological conditions re-shape physiological Tregs into pathological Tregs. Burns & Trauma, 2015. 3(1): p. 1.

2.       Xing, Y. and K.A. Hogquist, T-cell tolerance: central and peripheral. Cold Spring Harbor perspectives in biology, 2012. 4(6): p. a006957.

3.       Safinia, N., C. Scotta, T. Vaikunthanathan, R.I. Lechler, and G. Lombardi, Regulatory T cells: serious contenders in the promise for immunological tolerance in transplantation. Frontiers in immunology, 2015. 6.

4.       Ohkura, N., Y. Kitagawa, and S. Sakaguchi, Development and maintenance of regulatory T cells. Immunity, 2013. 38(3): p. 414-423.

5.       Ellis, G.I., M.C. Reneer, A.C. Vélez-Ortega, A. McCool, and F. Martí, Generation of induced regulatory T cells from primary human naïve and memory T cells. JoVE (Journal of Visualized Experiments), 2012(62): p. e3738-e3738.

6.       Schmitt, E.G. and C.B. Williams, Generation and function of induced regulatoryT cells. Spinal cord, 2012. 10(15).

7.       Baecher-Allan, C., V. Viglietta, and D.A. Hafler. Human CD4+ CD25+ regulatory T cells. in Seminars in immunology. 2004. Elsevier.

8.       Lastovicka, J., The phenotypic markers of CD4+ CD25+ T regulatory lymphocytes. Res Immunol, 2013: p. 1-14.

9.       Xie, X., M.J. Stubbington, J.K. Nissen, K.G. Andersen, D. Hebenstreit, S.A. Teichmann, et al. The regulatory T Cell lineage factor Foxp3 regulates gene expression through several distinct mechanisms mostly independent of direct DNA binding. PLoS Genet, 2015. 11(6): p. e1005251.

10.     Fontenot, J.D., M.A. Gavin, and A.Y. Rudensky, Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nature immunology, 2003. 4(4): p. 330-336.

11.     Charbonnier, L.-M., E. Janssen, J. Chou, T.K. Ohsumi, S. Keles, J.T. Hsu,et al.G. Dbaibo, Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked–like disorder caused by loss-of-function mutations in LRBA. Journal of Allergy and Clinical Immunology, 2015. 135(1): p. 217-227. e9.

12.     Chen, Q., Y.C. Kim, A. Laurence, G.A. Punkosdy, and E.M. Shevach, IL-2 controls the stability of Foxp3 expression in TGF-β–induced Foxp3+ T cells in vivo. The Journal of Immunology, 2011. 186(11): p. 6329-6337.

13.     Nguyen, T.-L.M., N.T. Makhlouf, B.A. Anthony, R.M. Teague, and R.J. DiPaolo, In vitro induced regulatory T cells are unique from endogenous regulatory T cells and effective at suppressing late stages of ongoing autoimmunity. PloS one, 2014. 9(8): p. e104698.

14.     HOPE, A.S.F.O., Foxp3: Important regulatory gene for the development of regulatory T-cells.

15.     Rossetti, M., R. Spreafico, S. Saidin, C. Chua, M. Moshref, J.Y. Leong,et al.S. Albani, Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region. The Journal of Immunology, 2015. 194(1): p. 113-124.

16.     Allan, S.E., L. Passerini, R. Bacchetta, N. Crellin, M. Dai, P.C. Orban,et al.M.K. Levings, The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs. The Journal of clinical investigation, 2005. 115(11): p. 3276-3284.

17.     Ha, M. and V.N. Kim, Regulation of microRNA biogenesis. Nature reviews Molecular cell biology, 2014. 15(8): p. 509-524.

18.     Chen, C.Z., S. Schaffert, R. Fragoso, and C. Loh, Regulation of immune responses and tolerance: the microRNA perspective. Immunological reviews, 2013. 253(1): p. 112-128.

19.     Sharma, V.K., S.V. Kaveri, and J. Bayry, Impaired regulatory T cell function in autoimmune diseases: are microRNAs the culprits&quest. Cellular & molecular immunology, 2015.

20.     Deng, X., Y. Su, H. Wu, R. Wu, P. Zhang, Y. Dai,et al.Q. Lu, The role of microRNAs in autoimmune diseases with skin involvement. Scandinavian journal of immunology, 2015. 81(3): p. 153-165.

21.     Fayyad-Kazan, H., R. Rouas, M. Fayyad-Kazan, R. Badran, N. El Zein, P. Lewalle,et al.M. Merimi, MicroRNA profile of circulating CD4-positive regulatory T cells in human adults and impact of differentially expressed microRNAs on expression of two genes essential to their function. Journal of Biological Chemistry, 2012. 287(13): p. 9910-9922.

22.     Okoye, I.S., S.M. Coomes, V.S. Pelly, S. Czieso, V. Papayannopoulos, T. Tolmachova,et al.M.S. Wilson, MicroRNA-containing T-regulatory-cell-derived exosomes suppress pathogenic T helper 1 cells. Immunity, 2014. 41(1): p. 89-103.

23.     Rouas, R., H. Fayyad‐Kazan, N. El Zein, P. Lewalle, F. Rothé, A. Simion,et al.A. Burny, Human natural Treg microRNA signature: role of microRNA‐31 and microRNA‐21 in FOXP3 expression. European journal of immunology, 2009. 39(6): p. 1608-1618.

24.     Grant, C.R., R. Liberal, G. Mieli-Vergani, D. Vergani, and M.S. Longhi, Regulatory T-cells in autoimmune diseases: challenges, controversies and—yet—unanswered questions. Autoimmunity reviews, 2015. 14(2): p. 105-116.

25.     Carbone, F., V. De Rosa, P.B. Carrieri, S. Montella, D. Bruzzese, A. Porcellini,et al.G. Matarese, Regulatory T cell proliferative potential is impaired in human autoimmune disease. Nature medicine, 2014. 20(1): p. 69-74.

26.     Long, S.A. and J.H. Buckner, CD4+ FOXP3+ T regulatory cells in human autoimmunity: more than a numbers game. The Journal of Immunology, 2011. 187(5): p. 2061-2066.

27.     Grimson, A., K.K.-H. Farh, W.K. Johnston, P. Garrett-Engele, L.P. Lim, and D.P. Bartel, MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Molecular cell, 2007. 27(1): p. 91-105.

28.     Saki, N., S. Abroun, M. Soleimani, S. Hajizamani, M. Shahjahani, R.E. Kast, and Y. Mortazavi, Involvement of microRNA in T-cell differentiation and malignancy. International journal of hematology-oncology and stem cell research, 2015. 9(1): p. 33.

29.     Simpson, L.J. and K.M. Ansel, MicroRNA regulation of lymphocyte tolerance and autoimmunity. Journal of Clinical Investigation, 2015. 125(6): p. 2242.

30.     Cobb, B.S., A. Hertweck, J. Smith, E. O'Connor, D. Graf, T. Cook,et al. A role for Dicer in immune regulation. The Journal of experimental medicine, 2006. 203(11): p. 2519-2527.

31.     Zhou, X., L.T. Jeker, B.T. Fife, S. Zhu, M.S. Anderson, M.T. McManus, ,et al. Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity. The Journal of experimental medicine, 2008. 205(9): p. 1983-1991.

32.     Barbi, J., D. Pardoll, and F. Pan, Treg functional stability and its responsiveness to the microenvironment. Immunological reviews, 2014. 259(1): p. 115-139.

33.     Dong, L., X. Wang, J. Tan, H. Li, W. Qian, J. Chen,et al.C. Tao, Decreased expression of microRNA‐21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis. Journal of cellular and molecular medicine, 2014. 18(11): p. 2213-2224.

34.     Yao, Q., S. Cao, C. Li, A. Mengesha, B. Kong, and M. Wei, Micro‐RNA‐21 regulates TGF‐β‐induced myofibroblast differentiation by targeting PDCD4 in tumor‐stroma interaction. International Journal of Cancer, 2011. 128(8): p. 1783-1792.

35.     Monfared, H., S.A.M. Ziaee, M. Hashemitabar, H. Khayatzadeh, V. Kheyrollahi, M. Tavallaei, and S.J. Mowla, Co-Regulated Expression of TGF-[Beta] Variants and miR-21 In Bladder Cancer. Urology journal, 2013. 10(3): p. 981.

36.     Liu, C., B. Li, Y. Cheng, J. Lin, J. Hao, S. Zhang,et al.L. Zhao, MiR-21 plays an important role in radiation induced carcinogenesis in BALB/c mice by directly targeting the tumor suppressor gene Big-h3. Int J Biol Sci, 2011. 7(3): p. 347-63.

37.     Qian, B., D. Katsaros, L. Lu, M. Preti, A. Durando, R. Arisio,et al.H. Yu, High miR-21 expression in breast cancer associated with poor disease-free survival in early stage disease and high TGF-β1. Breast cancer research and treatment, 2009. 117(1): p. 131-140.

38.     Sheedy, F.J., E. Palsson-McDermott, E.J. Hennessy, C. Martin, J.J. O'Leary, Q. Ruan,et al.L.A. O'Neill, Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21. Nature immunology, 2010. 11(2): p. 141-147.

39.     Das, A., K. Ganesh, S. Khanna, C.K. Sen, and S. Roy, Engulfment of apoptotic cells by macrophages: a role of microRNA-21 in the resolution of wound inflammation. The Journal of Immunology, 2014. 192(3): p. 1120-1129.

40.     Ma, X., S.N. Choudhury, L.B. Buscaglia, and Y. Li, The three-throng role of miR-21 in cancer. Cancer Research, 2012. 72(8 Supplement): p. 205-205.

41.     Gygi, S.P., Y. Rochon, B.R. Franza, and R. Aebersold, Correlation between protein and mRNA abundance in yeast. Molecular and cellular biology, 1999. 19(3): p. 1720-1730.

42.     Schwanhäusser, B., D. Busse, N. Li, G. Dittmar, J. Schuchhardt, J. Wolf,et al.M. Selbach, Global quantification of mammalian gene expression control. Nature, 2011. 473(7347): p. 337-342.

43.     Sharova, L.V., A.A. Sharov, T. Nedorezov, Y. Piao, N. Shaik, and M.S. Ko, Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells. DNA research, 2009. 16(1): p. 45-58.


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