Miyeloproliferatif Neoplazilerde JAK2V617F Mutasyonunun Endotel Hücresine Etkisi ve SOCS1-4 Gen Anlatımlarına Yansıması

Year 2020, Volume: 3 Issue: 3, 135 - 147, 05.11.2020

Ahmet Göksu Hilal Hekimoğlu Selçuk Sözer Tokdemir

Abstract

Amaç: Miyeloproliferatif neoplazi (MPN)’li hastalar morbidite ve mortalitesi yüksek tromboz riski taşırlar. Tromboza olan bu yatkınlık, artmış kan hücre sayısı ile bağdaştırılmış olsa da, endotel hücre (EH) fonksiyon bozukluğu gibi faktörler de önemli rol oynamaktadır. EH’nin miyeloproliferatif bozukluklarla olan bağlantısını göstermek üzere yapılan önceki çalışmalarda EH’nin MPN kan hücrelerinde %50-95 oranında mevcut olan JAK2V617F mutasyonunu taşıdığı gösterilmiş, ancak sitozolik tirozin kinaz olan Janus kinase 2 (JAK2) de meydana gelen bu mutasyonun EH üzerinde göstermiş olduğu morfolojik ve fonksiyonel değişiklik gösterilmemiştir. Ayrıca epigenetik faktörlerden Sitokin Sinyal Süpresör (SOCS) proteinleri‘nin MPN’deki etkisi de tam olarak bilinmemektedir. Bu çalışma ile JAK2V617F mutasyonunun EH'de yapmış olduğu etkileri epigenetik açıdan önemli olan, SOCS 1-4 gen anlatımları ve metilasyon durumları incelenmiştir. Gereç ve Yöntem: In vitro yeşil floresan proteini (GFP) ile işaretlenmiş JAK2 (yabanil formu) veya JAK2V617F mutasyonu taşıyan lentivirusün insan kordon veni endotel hücresi (HUVEC) içine transdüksiyon metodu kullanılmıştır. Genetik olarak modifiye edilmiş EH populasyonunda, akımölçer cihazı ile GFP tespit edilmiş ve analizleri yapılmıştır. Ardından hücre ayırıcı ile seçilmiş olan GFP+ hücrelerde DNA ve RNA izolasyonları gerçekleştirilmiştir. SOCS1-4 genlerinin anlatım değerleri gerçek zamanlı RT-PZR tespit edilmiş ve bisülfit modifikasyona maruz bırakılan DNA yardımıyla metilasyona özgü PZR yapılmıştır. Bulgular: JAK2, JAK2V617F ve GFP taşıyan lentiviral vektörlerle başarılı bir şekilde EH enfeksiyonu gerçekleştirilmiştir. Genetiği modifiye edilmiş EH'ler birbirleri ile kıyaslandığında morfolojik bir farklılık saptanmamıştır. SOCS1-4 genlerinde, mutant EH de JAK2 ve GFP'e göre gen anlatım düzeyinde anlamlı artış tespit edilmiştir. GFP ve JAK2 de tespit edilen SOCS1-4 gen anlatım baskılanmasında bu genlerde oluşması muhtemel bir metilasyon olmadığı tespit edilmiştir. Sonuç: Bu konuda gelecekte yapılacak çalışmalar, JAK2V617F’in EH’ler üzerindeki etkilerini ve trombotik olaylarla bağlantılısını açıklamamıza yardımcı olacaktır.

Keywords

Miyeloproliferatif Neoplazi (MPN), JAK2V617F, Endotel Hücre, Lentivirüs, SOCS

Supporting Institution

İstanbul Üniversitesi Bilimsel Araştırma Projeleri Birimi (BAP) tarafından desteklenmiştir.

Project Number

Proje No: 27570

The effect of JAK2V617F Mutation to the Endothelial Cells and the Expression Profiles of SOCS1-4 Genes in Myeloproliferative Neoplasms

Abstract

Objective: Patients with myeloproliferative neoplasms (MPN) are at an increased risk of developing thrombosis which leads to significant levels of morbidity and mortality. Although this propensity for thrombosis has been attributed to increased blood counts, other factors such as endothelial cell (EC) dysfunction likely play an important role. Previous studies have provided evidence of EC involvement by JAK2V617F which is known to be present in blood cells of 50-95% of patients with MPN. Furthermore, the morphological and functional changes of this mutation, a single nucleotide mutation in the cytosolic tyrosine kinase, Janus kinase 2 (JAK2) has not been shown in EC. In addition, the role of epigenetic factors including suppressors of cytokine signaling (SOCS) proteins is also unknown for MPN. In this study, the effects of the JAK2V617F mutation on EC and the SOCS 1-4 genes expression and their methylation profiles were investigated. Materials and Methods: The in vitro transduction method of green fluorescent protein (GFP) labeled lentivirus carrying the JAK2 (wild form) or JAK2V617F mutation into human cord vein endothelial cell (HUVEC) was applied. GFP was determined and analyzed with the flow cytometer. Afterwards, the genetically modified EC population was isolated, gating for GFP signal by cell sorter and then DNA and RNA isolations were performed. Gene expressional changes of SOCS1-4 genes were determined in real time RT-PCR and methylation specific PCR was performed with bisulfite modification of DNA. Results: The successful infection of JAK2, JAK2V617F and GFP vectors into EC were achieved. The comparison of the genetically modified EC revealed no morphological differences. A significant increase in the expressions of SOCS1-4 were detected in mutant EH compared to JAK2 and GFP. It was determined that methylation had no effect on suppression of SOCS1-4 expression detected in GFP and JAK2 EC. Conclusion: Future studies will further enlighten the effects of JAK2V617F on EC and its role in thrombotic events.

Keywords

Myeloroliferative Neoplasm (MPN), JAK2V617F, Endothelial Cells, Lentivirus, SOCS

Project Number

Proje No: 27570

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