Hücre Serilerinde Kilitli Nükleik Asitler İle MIR223 Gen Sessizleştirmesi

Year 2020, Volume: 3 Issue: 2, 45 - 50, 10.07.2020

Orçun Taşar , Müge Sayitoğlu , Özden Hatırnaz Ng

Abstract

Amaç: Kodlama yapmayan küçük RNA’lar hücre farklılaşması, büyümesi, gelişmesi, immün reaksiyonlar, stres adaptasyonu gibi fizyolojik süreçlerin yanı sıra, kanser, kalp hastalıkları gibi kompleks hastalıklarla da ilişkilendirilmiştir. MIR223, hematopoetik sisteme özgü bir miRNA’dır. T-hücreli Akut Lenfoblastik Lösemi (T-ALL) patogenezine katkıda bulunan miRNA’lar arasında yüksek anlatıma sahip olduğu ve onkomir olarak aktivite gösterdiği tespit edilmiştir. Bu çalışmada MIR223 geninin T-ALL hücre serilerinde alternatif bir yaklaşım olarak özgün kilitli nükleik asit (KNA) kullanılarak baskılanması ve gen sessizleştirme etkinliğinin gösterilmesi amaçlanmıştır. Gereç ve Yöntem: Kültüre edilen T-ALL hücre serilerine (Jurkat ve Molt4), 24 ve 48 saatlik sürelerde, 100 ve 150pmol konsantrasyonda MIR223’e özgü KNA uygulanmıştır. Her iki zaman aralığında RNA izolasyonu sonrası, stem loop polimeraz zincir reaksiyonu (PZR) ile cDNA sentezlenerek, kantitatif gerçek zamanlı PZR (QRT-PZR) ile miRNA anlatım düzeyleri belirlenmiştir. Bulgular: Her iki hücre serisinde de 24. saatte, 150pmol KNA, sadece transfeksiyon ajanı uygulanmış (Mock) hücrelerle karşılaştırıldığında MIR223 düzeyinin yüksek oranda baskılandığı gözlenmiştir (Jurkat %73, p=0,001 ve Molt4%80 p=0,04). Molt 4 hücre serisinde anlamlı düzeyde baskılanma 48. saatte devam etse de (p=0,005), Jurkat hücre serisinde 48. saatteki baskılama istatistiksel olarak anlamlı bulunmamıştır. Sonuç: MIR223 onkogenik etki gösteren bir miRNA olarak tanımlanmıştır ve antisens oligolar ile MIR223 geninin sessizleştirilmesi T-ALL gibi artmış MIR223 anlatımı gösteren kanserlerde hastalığın seyrini ve tedavi alternatiflerini araştırma imkanı sağlamaktadır. Bu çalışmada T-ALL hücrelerinde alternatif bir RNA interferans (sessizleştirme) uygulaması olarak KNA kullanılmıştır ve bu moleküllerin çok etkin ancak kısa süreli olarak kullanılabileceği görülmüştür.

Keywords

T-ALL, MIR223, kilitli nükleik asitler (KNA), gen sessizleştirmesi

Supporting Institution

Bu çalışma, İstanbul Üniversitesi Bilimsel Araştırma Projeleri Birimi tarafından desteklenmiştir.

Project Number

40487

MIR223 Gene Silencing via Locked Nucelic Acids in Cell Lines

Abstract

Objective: Noncoding small RNAs play roles in physiological processes such as cell differentiation, growth, development, immune reactions, stress adaptation as well as complex diseases such as cancer and heart diseases. MIR223 is a specific miRNA to the hematopoietic system. Among the miRNAs associated with the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), MIR223 was shown to have the highest expression level and functions as an oncomir. In this study we aimed to suppress MIR223 gene expression by using locked nucleic acid (LNA) in T-ALL cell lines as an alternative gene silencing technique and to show the silencing efficacy. Material and Methods: T-ALL cell lines (Jurkat and Molt4) were cultured and MIR223 specific LNA was applied in 100pmol and 150pmol concentrations for 24 hours and 48 hours. RNA was isolated from cells at both time points, followed by a stem loop polymerase chain reaction (PCR) for cDNA synthesis and the miRNA expression levels were evaluated by quantitative real time PCR. Results: In both cell lines a 150pmol application of LNA was compared to mock cells and the MIR223 expression was already suppressed at 24hours (73% in Jurkat (p=0,001) and 80%in Molt4 (p=0,04)). The suppression was continued at 48 hours in Molt4 cell line (p=0,005) where as there was no statistical significant difference in Jurkat at 48 hours. Conclusion: MIR223 has been identified as an oncomir and silencing the MIR223 with synthetic antisense oligos provide the opportunity to investigate the course and alternative therapy options of cancers with increased MIR223 expression, such as T-ALL. In this study LNA was used as an alternative RNA interference application in T-ALL cells and showed that LNA can effectively suppress gene expression when used in the short term. 

Keywords

T-ALL, MIR223, locked nucleic acid (LNA), Gene silencing

Project Number

40487

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