Published date: Sep 30 2024

Journal Title: Sudan Journal of Medical Sciences

Issue title: Sudan JMS: Volume 19 (2024), Issue No. 3

Pages: 389 – 400

DOI: 10.18502/sjms.v19i3.16171

Shirin Assarsh758us@yahoo.comClinical Research Development Center, Imam Reza Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran

Seyed Askar Roghaniaskar.roghani@gmail.comClinical Research Development Center, Imam Reza Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran

Clinical Research Development Center, Imam Reza Hospital, Kermanshah University of Medical Sciences, Kermanshah, Iran

Ramin Lotfiramin.lotfi1370@gmail.comBlood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Seyedeh Zahra Shahrokhvandshahrokhvand.zahra66@gmail.comImmunology Department, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran

Bahareh Kardidehbahare.kardide1990@gmail.comMedical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

Kheirollah Yarikyari@kums.ac.irMedical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

Bijan Soleymanibijan.soleymani@kums.acMedical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

Fatemeh Khademifkhademi60@gmail.comMedical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran

Background: The expression of forkhead box O (FOXO) was found to be connected with developing rheumatoid arthritis (RA), an inflammatory autoimmune disorder. The current study is intended to assess the expression and methylation status of the FOXO1 gene in individuals with recently diagnosed RA, before and after the administration of customary disease-modifying antirheumatic drugs (DMARDs).
Methods: Twenty participants were investigated in this study. The assessment of the FOXO1 gene expression in peripheral blood was done by real-time PCR, and the status of FOXO1 promoter methylation was ascertained via quantitative methylation-specific PCR (Q-MSP) before and after the administration of DMARDs for six months.
Results: Following DMARDs treatment, the study discovered a decrease in FOXO1 gene expression. However, the decline did not meet the criteria for statistically meaningful (P = 0.087). The expression of the FOXO1 gene was positively correlated with RA disease activity pre- and post-treatment with DMARDs (P = 0.009, r = 0.567 and P = 0.001, r = 0.656, respectively). Moreover, the study showed no alterations in the amount of DNA methylation of the FOXO1 promoter in newly diagnosed RA patients who had not yet received DMARDs, as compared to DMARDs-treated RA patients.
Conclusion: Altogether, this study suggests that DMARDs treatment may reduce FOXO1 gene expression, potentially helping to alleviate the pro-inflammatory effects associated with this gene.

Keywords: antirheumatic agents, disease activity, FOXO1, rheumatoid arthritis

[1] Yarwood, A., Huizinga, T. W. J., & Worthington, J. (2016). The genetics of rheumatoid arthritis: Risk and protection in different stages of the evolution of RA. Rheumatology (Oxford, England), 55(2), 199–209. https://doi.org/10.1093/rheumatology/keu323

[2] Lin, Y.-J., Anzaghe, M., & Schülke, S. (2020). Update on the pathomechanism, diagnosis, and treatment options for rheumatoid arthritis. Cells, 9(4), 880. Advance online publication. https://doi.org/10.3390/cells9040880

[3] Eijkelenboom, A., & Burgering, B. M. T. (2013). FOXOs: Signalling integrators for homeostasis maintenance. Nature Reviews. Molecular Cell Biology, 14, 83–97. https://doi.org/10.1038/nrm3507

[4] Wang, M., Zhang, X., Zhao, H., Wang, Q., & Pan, Y. (2009). FoxO gene family evolution in vertebrates. BMC Evolutionary Biology, 9, 222. https://doi.org/10.1186/1471-2148-9-222

[5] Kuo, C.-C., & Lin, S.-C. (2007). Altered FOXO1 transcript levels in peripheral blood mononuclear cells of systemic lupus erythematosus and rheumatoid arthritis patients. Molecular Medicine (Cambridge, Mass.), 13, 561–566. https://doi.org/10.2119/2007- 00021.Kuo

[6] Hosaka, T., Biggs, W. H., III, Tieu, D., Boyer, A. D., Varki, N. M., Cavenee, W. K., & Arden, K. C. (2004). Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proceedings of the National Academy of Sciences of the United States of America, 101(9), 2975–2980. https://doi.org/10.1073/pnas.0400093101

[7] Furuyama, T., Kitayama, K., Shimoda, Y., Ogawa, M., Sone, K., Yoshida-Araki, K., Hisatsune, H., Nishikawa, S., Nakayama, K., Nakayama, K., Ikeda, K., Motoyama, N., & Mori, N. (2004). Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice. The Journal of Biological Chemistry, 279(33), 34741– 34749. https://doi.org/10.1074/jbc.M314214200

[8] Accili, D., & Arden, K. C. (2004). FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell, 117(4), 421–426. https://doi.org/10.1016/S0092-8674(04)00452-0

[9] Luo, C. T., & Li, M. O. (2018). Foxo transcription factors in T cell biology and tumor immunity. Seminars in Cancer Biology, 50, 13–20. Elsevier. https://doi.org/10.1016/j.semcancer.2018.04.006

[10] Su, D., Coudriet, G. M., Hyun Kim, D., Lu, Y., Perdomo, G., Qu, S., Slusher, S., Tse, H. M., Piganelli, J., Giannoukakis, N., Zhang, J., & Dong, H. H. (2009). FoxO1 links insulin resistance to proinflammatory cytokine IL-1β production in macrophages. Diabetes, 58(11), 2624–2633. https://doi.org/10.2337/db09-0232

[11] Nwadozi, E., Roudier, E., Rullman, E., Tharmalingam, S., Liu, H. Y., Gustafsson, T., & Haas, T. L. (2016). Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet. The FASEB Journal, 30(9), 3039–3052. https://doi.org/10.1096/fj.201600245R

[12] Zhu, M., Goetsch, S. C., Wang, Z., Luo, R., Hill, J. A., Schneider, J., Morris, S. M., Jr., & Liu, Z.-P. (2015). FoxO4 promotes early inflammatory response upon myocardial infarction via endothelial Arg1. Circulation Research, 117(11), 967–977. https://doi.org/10.1161/CIRCRESAHA.115.306919

[13] Viatte, S., Plant, D., & Raychaudhuri, S. (2013). Genetics and epigenetics of rheumatoid arthritis. Nature Reviews. Rheumatology, 9, 141–153. https://doi.org/10.1038/nrrheum.2012.237

[14] Issa, J.-P. J., Ottaviano, Y. L., Celano, P., Hamilton, S. R., Davidson, N. E., & Baylin, S. B. (1994). Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nature Genetics, 7, 536–540. https://doi.org/10.1038/ng0894-536

[15] Esteller, M. (2002). CpG island hypermethylation and tumor suppressor genes: A booming present, a brighter future. Oncogene, 21, 5427–5440. https://doi.org/10.1038/sj.onc.1205600

[16] Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes & Development, 16, 6– 21. https://doi.org/10.1101/gad.947102

[17] Vihinen, M., & Mäntsälä, P. (1989). Microbial amylolytic enzymes. Critical Reviews in Biochemistry and Molecular Biology, 24(4), 329–418. https://doi.org/10.3109/10409238909082556

[18] Aslani, S., Mahmoudi, M., Karami, J., Jamshidi, A. R., Malekshahi, Z., & Nicknam, M. H. (2016). Epigenetic alterations underlying autoimmune diseases. Autoimmunity, 49(2), 69–83. https://doi.org/10.3109/08916934.2015.1134511

[19] Liu, C.-C., Fang, T.-J., Ou, T.-T., Wu, C.-C., Li, R.-N., Lin, Y.-C., Lin, C.-H., Tsai, W.-C., Liu, H.- W., & Yen, J.-H. (2011). Global DNA methylation, DNMT1, and MBD2 in patients with rheumatoid arthritis. Immunology Letters, 135(1–2), 96–99. https://doi.org/10.1016/j.imlet.2010.10.003

[20] Miao, C. G., Yang, Y. Y., He, X., & Li, J. (2013). New advances of DNA methylation and histone modifications in rheumatoid arthritis, with special emphasis on MeCP2. Cellular Signalling, 25(4), 875– 882. https://doi.org/10.1016/j.cellsig.2012.12.017

[21] Glossop, J. R., Emes, R. D., Nixon, N. B., Packham, J. C., Fryer, A. A., Mattey, D. L., & Farrell, W. E. (2016). Genome-wide profiling in treatment-naive early rheumatoid arthritis reveals DNA methylome changes in T and B lymphocytes. Epigenomics, 8(2), 209–224. https://doi.org/10.2217/epi.15.103

[22] de Andres, M. C., Perez-Pampin, E., Calaza, M., Santaclara, F. J., Ortea, I., Gomez-Reino, J. J., & Gonzalez, A. (2015). Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate. Arthritis Research & Therapy, 17, 233. https://doi.org/10.1186/s13075-015-0748-5

[23] Brown, P. M., Pratt, A. G., & Isaacs, J. D. (2016). Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers. Nature Reviews. Rheumatology, 12, 731–742. https://doi.org/10.1038/nrrheum.2016.175

[24] Wang, Y.-C., & Chiang, E.-P. I. (2012). Lowdose methotrexate inhibits methionine Sadenosyltransferase in vitro and in vivo. Molecular Medicine (Cambridge, Mass.), 18, 423–432. https://doi.org/10.2119/molmed.2011.00048

[25] Nesher, G., & Moore, T. L. (1990). The in vitro effects of methotrexate on peripheral blood mononuclear cells. Modulation by methyl donors and spermidine. Arthritis and Rheumatism, 33(7), 954–959. https://doi.org/10.1002/art.1780330706

[26] Kim, Y. I., Logan, J. W., Mason, J. B., & Roubenoff, R. (1996). DNA hypomethylation in inflammatory arthritis: Reversal with methotrexate. The Journal of Laboratory and Clinical Medicine, 128(2), 165–172. https://doi.org/10.1016/S0022-2143(96)90008-6

[27] Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29(9), e45. https://doi.org/10.1093/nar/29.9.e45

[28] Miller, S. A., Dykes, D. D., & Polesky, H. F. (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16(3), 1215. https://doi.org/10.1093/nar/16.3.1215

[29] Li, L.-C., & Dahiya, R. (2002). MethPrimer: Designing primers for methylation PCRs. Bioinformatics (Oxford, England), 18(11), 1427–1431. https://doi.org/10.1093/bioinformatics/18.11.1427

[30] Carlsson, P., & Mahlapuu, M. (2002). Forkhead transcription factors: Key players in development and metabolism. Developmental Biology, 250(1), 1– 23. https://doi.org/10.1006/dbio.2002.0780

[31] Kaestner, K. H., Knöchel, W., & Martínez, D. E. (2000). Unified nomenclature for the winged helix/forkhead transcription factors. Genes & Development, 14, 142– 146. https://doi.org/10.1101/gad.14.2.142

[32] Burgering, B. M. T., & Kops, G. J. P. L. (2002). Cell cycle and death control: Long live Forkheads. Trends in Biochemical Sciences, 27(7), 352–360. https://doi.org/10.1016/S0968-0004(02)02113-8

[33] Fontenot, J. D., Gavin, M. A., & Rudensky, A. Y. (2003). Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nature Immunology, 4, 330–336. https://doi.org/10.1038/ni904

[34] Grabiec, A. M., Angiolilli, C., Hartkamp, L. M., van Baarsen, L. G. M., Tak, P. P., & Reedquist, K. A. (2015). JNK-dependent downregulation of FoxO1 is required to promote the survival of fibroblast-like synoviocytes in rheumatoid arthritis. Annals of the Rheumatic Diseases, 74(9), 1763–1771. https://doi.org/10.1136/annrheumdis-2013-203610

[35] Lin, Y., & Luo, Z. (2017). Aberrant methylation patterns affect the molecular pathogenesis of rheumatoid arthritis. International Immunopharmacology, 46, 141–145. https://doi.org/10.1016/j.intimp.2017.02.008

[36] Karouzakis, E., Gay, R. E., Michel, B. A., Gay, S., & Neidhart, M. (2009). DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis and Rheumatism, 60(12), 3613–3622. https://doi.org/10.1002/art.25018

[37] Nakano, K., Whitaker, J. W., Boyle, D. L., Wang, W., & Firestein, G. S. (2013). DNA methylome signature in rheumatoid arthritis. Annals of the Rheumatic Diseases, 72(1), 110–117. https://doi.org/10.1136/annrheumdis-2012-201526

[38] Karami, J., Aslani, S., Tahmasebi, M. N., Mousavi, M. J., Sharafat Vaziri, A., Jamshidi, A., Farhadi, E., & Mahmoudi, M. (2020). Epigenetics in rheumatoid arthritis; Fibroblast-like synoviocytes as an emerging paradigm in the pathogenesis of the disease. Immunology and Cell Biology, 98(3), 171– 186. https://doi.org/10.1111/imcb.12311

[39] Gosselt, H. R., van Zelst, B. D., de Rotte, M. C. F. J., Hazes, J. M. W., de Jonge, R., & Heil, S. G. (2019). Higher baseline global leukocyte DNA methylation is associated with MTX non-response in early RA patients. Arthritis Research & Therapy, 21, 157. https://doi.org/10.1186/s13075-019-1936-5