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Mediterr J Rheumatol 2018;29(1):56-8
Role of miR200b-5p miRNA in lymphomagenesis associated with Sjögren’s syndrome (SS)
Authors Information

Department of Pathophysiology, School of Medicine, National and Kapodistrian University of Athens, Greece

Abstract

Background: Development of non-Hodgkin’s lymphoma (NHL) is the major adverse outcome of primary Sjögren’s Syndrome (pSS) affecting both morbidity and mortality. The high frequency of transformation to lymphoid malignancy in pSS among autoimmune rheumatic diseases (6-10% of patients) and the accessibility of the affected organ (minor salivary glands; MSG), render pSS an ideal model for the study of lymphomagenesis associated with autoimmune diseases and inflammation. Although pSS-related lymphoid transformation is generally considered as an antigen-driven, multi-step process owed to the chronic activation of B-cells in MSGs, the underlying mechanisms remain elusive. Our recent results support that miR200b-5p miRNA is significantly down-regulated in the MSGs of pSS patients who have or will develop lymphoma, long before lymphoma clinical onset, indicating that it may be involved in lymphomagenesis. Aim: To investigate the role of miR200b-5p miRNA in pSS-associated lymphomagenesis. Methods: At first, the miR200b-5p-expression will be examined by in situ hybridization in MSGs of pSS patients who are at low risk and have not developed NHL during follow-up, high risk and developed NHL in the future (pre-lymphoma) or have NHL, and the expressing cellular types, as well as those with reduced expression during lymphomagenesis, will be identified. Then, the miR200b-5p targeted molecular pathways in those cellular types (epithelial, B-cells and/or other lymphocytes, all non-neoplastic) will be studied in in vitro experiments by over-expressing and silencing of miR200b-5p, followed by transcriptome analysis. This approach is expected to find possibly novel pathogenetic mechanisms underlying SS-related lymphomagenesis. The latter is of high significance, not only for the understanding of lymphomagenesis, but also for its reversal and/or treatment. Anticipated Benefits: This approach is anticipated to a) reveal the differentially regulated molecules and pathways by miR200b-5p, b) enlighten novel pathogenetic pathways underlying lymphomagenesis and c) identify novel therapeutic targets and possibly evidence-based therapeutic interventions.  

https://doi.org/10.31138/mjr.29.1.56

Full Text

BACKGROUND/INTRODUCTION

The development of B-cell non-Hodgkin lymphoma (NHL) is common in primary Sjögren’s syndrome (pSS) (6-10% of patients) and affects both morbidity and mortality.1-6 In the majority of pSS patients, NHL starts in the exocrine glands, such as the salivary glands, which are the major target of pSS autoimmune responses. The high frequency of transformation to lymphoid malignancy in pSS among autoimmune rheumatic diseases7,8 and the accessibility of the affected organ, the minor salivary glands (MSG), render pSS an ideal model for the study of lymphomagenesis associated with autoimmune diseases and inflammation. The mechanisms underlying the development of neoplasia in pSS remain elusive. The existence of several clinical, laboratory and histological features present at diagnosis, including salivary gland enlargement (SGE), purpura, vasculitis, leukopenia, cryoglobulinemia, hypocomplementemia, rheumatoid factor, the severity of histopathologic MSG infiltrates, their organization into germinal centers (GC) and the infiltration by certain cell types, such as macrophages, in the pSS patients who are at high-risk to develop NHL in the future3,9-15 suggest that it is a chronic, multi-step process. Generally, it is considered that lymphomagenesis in pSS is a multistep process that arises from the chronic, continuous, antigen-driven B-cell stimulation resulting in the ineffective control of IgV gene recombination, chromosomal translocations, activation of proto-oncogenes, inactivation of tumor-suppressor genes, and ultimately to malignant transformation.14,16 The cellular source of the antigenic stimulation of B cells and the type of antigens are elusive. The organization of MSG infiltrates in ectopic germinal centers (GC) is considered critical for the activation of autoreactive B cells and the development of MALT lymphomas.15,17,18 Furthermore, salivary gland epithelial cells (SGECs) that are the key regulators of the pSS autoimmune responses in MSGs,19,20 have been shown to drive the differentiation of B cells, in a similar manner to that observed in MSG lesions,19,21 suggesting that they may be involved in the chronic activation of B cells and lymphomagenesis. The molecular pathways underlying the lymphomagenesis in pSS are under study.

Our recent evidence supports that miR200b-5p may be implicated in the lymphomagenesis associated with pSS. Although not de-regulated compared to sicca-controls, the expression levels of miR200b-5p in the MSGs were significantly reduced in pSS patients who were going to develop or had NHL compared to those who were at low-risk for developing lymphoma and did not had lymphoma during follow-up (22 and unpublished data). This decrease was evident long before the clinical onset of lymphoma, whereas the study of paired sequential MSG specimens before and on lymphoma diagnosis showed that the low miR200b-5p levels remained rather stable through transformation to lymphoma. These findings support that miR200b-5p may have a pathogenetic role in pSS-associated lymphomagenesis. Little is known for miR200b-5p, possibly because it represents the star strand, which is generally considered to degrade during miRNA biogenesis. Recently, it has been reported that miR200b-5p controls the non-canonical EMT in synergy with miR200b-3p by targeting PRKCA and PIP4K2A molecules in the RHOGDI pathway.23 However, the miR200b miRNAs, mainly miR200b-3p, are thought central regulators of oncogenesis, tumor metastasis and drug resistance of solid tumors by regulating epithelial-to-mesenchymal transition (EMT) primarily by down-regulation of the zinc-finger E-box-binding homeobox-1 (ZEB1) transcription factor and AKT activation.24,25 Furthermore, elevated miR200b expression and subsequent inhibition of ZEB1 transcription factor and increased BCL6 protein expression has been associated with better prognosis of the Helicobacter pylori-positive gastric diffuse large B-cell lymphomas, compared to Helicobacter pylori-negative ones.26

 

AIM OF THE STUDY

Our findings support the implication of miR200b-5p miRNA in the lymphomagenesis in pSS. In this study, we propose the dissection of its role by identifying the expressing cells and the regulated molecular pathways.

 

RESEARCH PLAN – METHODS

The cells expressing miR200b-5p in the MSG tissues, as well as those with reduced expression in MSGs from pre-lymphoma and lymphoma pSS patients, will be identified by in situ hybridization (ISH) using commercially available LNA-probes (Exiqon). Subsequently, the function of miR200b-5p in these cell types (SGEC, B, T and/or other type of lymphocytes) will be interrogated by silencing and overexpression in vitro experiments using specific miRNA inhibitors and mimics, respectively, followed by transcriptome analysis for the identification of the regulated genes and pathways. Finally, the pathogenetic potential of the identified pathways in the lymphomagenesis of pSS will be verified by investigating the expression of the molecules comprising the pathway in MSG tissues, SGECs and B cells from pSS patients with lymphoma or not. The expression of these molecules will be performed at the mRNA level by qPCR and at the protein level by immunohistochemistry in MSG tissues and/or immunoblotting and flow cytometry in cultured non-neoplastic cells (SGECs, B, T and/or other type of lymphocytes isolated from the peripheral blood of healthy donors).

The proposed study has been approved by the Ethics Committee of School of Medicine, NKUA, Greece (Protocol-No.: 1516023881).
 

IMPACT OF THE STUDY

The study is expected to a) reveal the differentially regulated molecules and pathways by miR200b-5p, b) enlighten novel pathogenetic pathways with therapeutic potential and c) facilitate the better understanding of the mechanisms underlying lymphomagenesis in pSS, which is mandatory for the discovery of novel therapeutic targets and/or the evidence-based therapeutic administration of existing agents. The study of the pathogenetic mechanisms underlying progression to lymphoma in pSS is mandatory, since it may facilitate the arrest of the progression to and/or the effective treatment of malignancy in pSS, as well as other lymphoid malignancies, as suggested by the low MSG expression of miR200b-5p in a patient with HBV-sialadenitis and MALT lymphoma (our unpublished data).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

References
  1. Tzioufas A G, Kapsogeorgou E K, Moutsopoulos H M. Pathogenesis of Sjogren's syndrome: what we know and what we should learn. J Autoimmun 2012;39:4-8.
  2. Voulgarelis M, Dafni U G, Isenberg D A, Moutsopoulos H M. Malignant lymphoma in primary Sjogren's syndrome: a multicenter, retrospective, clinical study by the European Concerted Action on Sjogren's Syndrome. Arthritis Rheum 1999;42:1765-72.
  3. Skopouli F N, Dafni U, Ioannidis J P, Moutsopoulos H M. Clinical evolution, and morbidity and mortality of primary Sjogren's syndrome. Semin Arthritis Rheum  2000;29:296-304.
  4. Theander E, Manthorpe R, Jacobsson L T. Mortality and causes of death in primary Sjogren's syndrome: a prospective cohort study. Arthritis Rheum 2004;50:1262-9.
  5. Papageorgiou A, Ziogas D C, Mavragani C P, et al. Predicting the outcome of Sjogren's syndrome-associated non-hodgkin's lymphoma patients. PloS one 2015;10(2):e0116189.
  6. Ioannidis J P, Vassiliou V A, Moutsopoulos H M. Long-term risk of mortality and lymphoproliferative disease and predictive classification of primary Sjogren's syndrome. Arthritis Rheum 2002;46:741-7.
  7. Kassan S S, Thomas T L, Moutsopoulos H M, et al. Increased risk of lymphoma in sicca syndrome. Ann Intern Med 1978;89:888-92.
  8. Zintzaras E, Voulgarelis M, Moutsopoulos H M. The risk of lymphoma development in autoimmune diseases: a meta-analysis. Arch Intern Med 2005;165:2337-44.
  9. Baimpa E, Dahabreh I J, Voulgarelis M, Moutsopoulos H M. Hematologic manifestations and predictors of lymphoma development in primary Sjogren syndrome: clinical and pathophysiologic aspects. Medicine 2009;88:284-93.
  10. Brito-Zeron P, Ramos-Casals M, Bove A, Sentis J, Font J. Predicting adverse outcomes in primary Sjogren's syndrome: identification of prognostic factors. Rheumatology 2007;46:1359-62.
  11. Christodoulou M I, Kapsogeorgou E K, Moutsopoulos H M. Characteristics of the minor salivary gland infiltrates in Sjogren's syndrome. J Autoimmun 2010;34:400-7.
  12. Gerli R, Muscat C, Giansanti M, et al. Quantitative assessment of salivary gland inflammatory infiltration in primary Sjogren's syndrome: its relationship to different demographic, clinical and serological features of the disorder. Br J Rheumatol 1997;36:969-75.
  13. Nocturne G, Virone A, Ng W F, et al. Rheumatoid Factor and Disease Activity Are Independent Predictors of Lymphoma in Primary Sjogren's Syndrome. Arthritis Rheum 2016;68:977-85.
  14. Papageorgiou A, Voulgarelis M, Tzioufas A G. Clinical picture, outcome and predictive factors of lymphoma in Sjogren syndrome. Autoimmun Rev 2015;14:641-9.
  15. Theander E, Vasaitis L, Baecklund E, et al. Lymphoid organisation in labial salivary gland biopsies is a possible predictor for the development of malignant lymphoma in primary Sjogren's syndrome. Ann Rheum Dis 2011;70:1363-8.
  16. Bombardieri M, Pitzalis C. Ectopic lymphoid neogenesis and lymphoid chemokines in Sjogren's syndrome: at the interplay between chronic inflammation, autoimmunity and lymphomagenesis. Curr Pharm Biotechnol 2012;13:1989-96.
  17. Bombardieri M, Barone F, Humby F, et al. Activation-induced cytidine deaminase expression in follicular dendritic cell networks and interfollicular large B cells supports functionality of ectopic lymphoid neogenesis in autoimmune sialoadenitis and MALT lymphoma in Sjogren's syndrome. J Immunol 2007;179:4929-38.
  18. Pitzalis C, Jones G W, Bombardieri M, Jones S A. Ectopic lymphoid-like structures in infection, cancer and autoimmunity. Nat Rev Immunol 2014;14:447-62.
  19. Kapsogeorgou E K, Tzioufas A G. Glandular epithelium: Innocent bystander or leading actor In: Roberto Gerli EB, Alessia Alunno, ed. Sjogren's Syndrome. Novel Insights in Pathogenic, Clinical and Therapeutic Aspects: Academic Press;2016:189-98.
  20. Moutsopoulos H M. Sjogren's syndrome: autoimmune epithelitis. Clin Immunol Immunopathol 1994;72:162-5.
  21. Morva A, Kapsogeorgou E K, Konsta O D, Moutsopoulos H M, Tzioufas A G. Salivary Gland Epithelial Cells (Sgecs) Promote the Differentiation of B Cells. Ann Rheum Dis 2013;72:77.
  22. Gourzi V C, Kapsogeorgou E K, Kyriakidis N C, Tzioufas A G. Study of microRNAs (miRNAs) that are predicted to target the autoantigens Ro/SSA and La/SSB in primary Sjogren's Syndrome. Clin Exp Immunol 2015;182:14-22.
  23. Rhodes L V, Martin E C, Segar H C, et al. Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer. Oncotarget Jun 30 2015;6:16638-52.
  24. Humphries B, Yang C. The microRNA-200 family: small molecules with novel roles in cancer development, progression and therapy. Oncotarget 2015;6:6472-98.
  25. Senfter D, Madlener S, Krupitza G, Mader R M. The microRNA-200 family: still much to discover. Biomol Concepts 2016;7:311-9.
  26. Huang W T, Kuo S H, Cheng A L, Lin C W. Inhibition of ZEB1 by miR-200 characterizes Helicobacter pylori-positive gastric diffuse large B-cell lymphoma with a less aggressive behavior. Mod Pathol 2014;27:1116-25.