MiR-34a-5p’NİN AŞIRI İFADE EDİLEN AML İLİŞKİLİ GENLER ÜZERİNDEKİ ETKİSİ

Amaç: Akut miyeloid lösemi (AML) ölümcül bir lösemi türüdür. AML ilişkili genlerin ekspresyonu sadece genetik değişikliklerle değil aynı zamanda mikroRNA’lar (miRNA’lar) gibi çeşitli epigenetik faktörlerle de değiştirilebilir. MiRNA’lar birçok genin ifade seviyesini değiştirerek hücrede oldukça kritik görevler yapabilmektedir. miRNA ve hedef genleri arasındaki etkileşimin tespit edilmesi, hastalığın moleküler mekanizmasının aydınlatılması açısından oldukça önemlidir. Çalışmamızda AML dahil birçok kanserde tümör baskılayıcı role sahip miR-34a-5p’nin AML hücre proliferasyonu üzerindeki etkisi ve AML ilişkili genlerin ifade değişimindeki rolü araştırılmıştır. Gereç ve Yöntem: Leukemia Gene and Literature Database web sitesi (http://soft.bioinfo-minzhao.org/lgl/)’de, lösemi ile ilişkili genleri içeren 600’den fazla AML ile ilgili gen bulunmaktadır. Bu web sayfasında yer alan miR-34a-5p’nin potansiyel hedef genlerini tanımlamak için yaptığımız bu çalışmada miRDB veri tabanı kullanılmıştır. Sonrasında miRWalk, miRTarbase, Tarbase ve miRNet araçlarıyla doğrulanmıştır. PPI etkileşimleri, yolak analizi, çeşitli biyoinformatik araçlar kullanılarak tanımlanmıştır. İn vitro olarak miR-34a-5p’nin AML hücreleri üzerindeki etkisi belirlenip NOTCH2, IGF1R, SKP2 ve CDC25A genlerinin mimik miR-34a-5p ile transfekte edilmiş NB4 ve HL60 hücrelerinde ekspresyonu araştırılmıştır. Bulgular: Çeşitli biyoinformatik araçlar kullanılarak miR-34a-5p tarafından hedeflenebilecek 44 AML ilişkili gen belirlenmiştir. Sonrasında yapılan in vitro çalışmada miR-34a-5p ile transfekte edilen hücrelerde proliferasyonun istatistiksel olarak anlamlı şekilde baskılandığı gözlenmiştir (48 saat HL60 hücreleri p=0,00011; NB4 hücreleri p=0,0031 ve 96 saat HL60 hücreleri p=0,00013; NB4 p=0,00018). miR-34a-5p mimik transfekte edilen NB4 ve HL60 hücrelerinde NOTCH2, IGF1R, SKP2 ve CDC25A mRNA ifade seviyelerinin kontrol gruplarına göre anlamlı şekilde azaldığı tespit edilmiştir (HL60 hücrelerinde sırasıyla (p=0,003; p=0,02; p=0,01; p=0,0009) ve NB4 hücrelerinde sırasıyla p=0,02; p=0,02; p=0,01; p=0,0007). Sonuç: miR-34a-5p; NOTCH2, IGF1R, SKP2 ve CDC25A gibi birçok geni hedefleyerek AML hücre proliferasyonunu inhibe edebilir. Bu genler ile miR-34a-5p arasındaki ilişkiyi net bir şekilde belirleyebilmek için daha ileri tekniklerle farklı çalışmaların yapılmasına ihtiyaç vardır. Çalışma sonuçlarımız, miRNA-hedef gen ilişkisi araştırılırken uygun biyoinformatik araçlarla in vitro yöntemlerin birlikte başarıyla kullanılabileceğini göstermektedir.

Anahtar Kelimeler:

miR-34a-5p, AML, NB4, HL60

THE EFFECT OF miR-34a-5p ON OVEREXPRESSED AML ASSOCIATED GENES

Objective: Acute myeloid leukemia (AML) is a deadly type of leukemia. The expression of AML-related genes may be altered not only by genetic changes but also by various epigenetic factors such as microRNAs (miRNAs). The expression levels of many genes can be altered by miRNAs. The detection of miRNA’s target genes is critical for an understanding of the disease’s molecular mechanism. In this study possible target genes of miR-34a-5p in AML were determined and the effect of the relationship between miR-34a-5p and target genes on the cancer process was investigated. Materials and Methods: Leukemia Gene and Literature Database web tool (http://soft.bioinfo-minzhao.org/lgl/) includes a useful leukemia gene and literature da. There are more than 600 AML-related genes on this database. In the present study, in order to define the potential target genes of miR-34a-5p on the database, we used miRDB tool and then confirmed the findings using miRWalk, miRTarbase, Tarbase and miRNet tools. Defined miR-34a-5p AML related genes were verified by the DisGeNET platform. A Protein-Protein Interaction (PPI) network analysis of the genes was conducted using several bioinformatics tools. The effect of miR-34a-5p on cell proliferation was investigated by transfecting mimic miR-34a-5p into HL60 and NB4 cells. The mRNA expressions of NOTCH2, IGF1R, SKP2 and CDC25A genes were investigated in miR-34a-5p transfected NB4 and HL60 cells and control groups. Results: Using bioinformatics tools we determined 44 AMLrelated genes that could be targeted by miR-34a-5p. According to our in vitro study results statistically significant suppression of proliferation was observed in miR-34a-5p transfected cells (48h HL60 cells p=0.00011; NB4 cells p=0.0031 and 96h HL60 cells p=0.00013; NB4 p=0.00018). It was also found that NOTCH2, IGF1R, SKP2 and CDC25A mRNA expressions were down regulated in miR-34a-5p mimic-transfected HL60 cells (p=0.003; p=0.02; p=0.01; p=0.0009 respectively) and NB4 cells (p=0.02; p=0.02; p=0.01; p=0.0007 respectively) compared to the control groups. Conclusion: miR-34a-5p may inhibit AML cell proliferation by targeting many genes like NOTCH2, IGF1R, SKP2 and CDC25A. The results of our study indicate that appropriate bioinformatics tools and in vitro methods can successfully be used together when investigating the relationship between miRNAs and target genes. Further studies are required to determine the detailed relationship between these genes and miR-34a-5p.

Keywords:

miR-34a-5p, AML, NB4, HL60,

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