Resveratrolün HL-60 Lösemi ve Raji Lenfoma Hücrelerinde Apoptozla Bağlantılı mikroRNA Profili Üzerindeki Etkisi

Year 2015, Volume: 16 Issue: 3, 83 - 90, 01.12.2015

Kemal Ergin Gökay Bozkurt Cankut Çubuk Safiye Aktaş

Abstract

Amaç: Bu çalışmanın amacı; HL-60 (promyelositik lösemi) ve Raji (Burkitt lenfoma) hücrelerinde, resveratrol uygulaması sonrası mikroRNA (miRNA) profillerinin belirlenmesi ve apoptoz oranlarının immünohistokimyasal metodlarla belirlenmesidir.Gereç ve Yöntemler: Deney dizaynı dört hücre kültürü grubu üzerinden yapılmıştır; 1. grup: HL-60 (Kontrol), 2. grup: HL-60 + resveratrol, 3. grup: Raji ve 4. grup: Raji + resveratrol. Ardından iki tekrarlı mikroarray analizi yapılmış, biyoenformatik analiz ile farklı miRNA ekspresyonları saptanmış ve sonrasında eş zamanlı polimeraz zincir reaksiyonu (EZ-PZR) ile validasyon yapılmıştır. Ayrıca TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) yöntemi ve immünohistokimyasal belirteçler ile apoptoz oranı belirlenmiştir.Bulgular: Mikroarray analizi sonrasında HL-60 (kontrol) ve HL-60 + resveratrol grupları arasında farklı eksprese olan sekiz adet miRNA (hsa-miR18a, hsa-let-7d, hsa-let-7b, hsa-miR-1246, hsa-miR-320b, hsa-miR-92a, hsa-miR-609 ve hsa-miR337-3p) saptanmıştır. Resveratrol verilen Raji grubunda kontrol grubuna kıyasla bir tane farklı miRNA (hsa-miR-378) eksprese olmuştur. Validasyon amacıyla yapılan EZ-PZR’de dört adet miRNA (miR-18a, miR-92a, miR-320a, miR-378) ile konfirmasyon gösterilmiştir. Kontrol grubuna kıyasla resveratrol verilen HL-60 grubunda apoptoz (TUNEL) oranı 2 kat daha yüksek olarak saptandı. Raji grubu ile resveratrol verilen Raji grubu arasında anlamlı bir fark saptanmamış olmakla birlikte resveratrol verilen grupta apoptoz oranında bir artma eğilimi saptandı. Bununla birlikte gruplar arasında immünohistokimyasal analizde özellikle Fas ve kaspaz belirteçleri daha belirgin olarak eksprese oldu. Sonuç: Bu çalışmada doğal bir biyoaktif madde olan resveratrolün HL-60 ve Raji hücrelerindeki etkisine bakılmış ve farklı miRNA ekspresyonları saptanmıştır. Ayrıca hücre ölümünün de özellikle kaspaz ve Fas mekanizması üzerinden olduğu düşünülmüştür. Bununla birlikte lösemi ve lenfomadaki olası terapötik potansiyelleri sebebiyle ileri fonksiyonel ve organizma çalışmalarının da yapılması gerekmektedir

Keywords

Resveratrol, mikroRNA, apoptoz, lösemi, lenfoma

Effect of Resveratrol on microRNA Profile and Apoptosis in HL-60 Leukemia and Raji Lymphoma Cells

Abstract

Objective: The purpose of this study was to determine the microRNA (miRNA) profiles and the apoptosis rates in the HL-60 (promyelocytic leukemia) and Raji (Burkitt’s lymphoma) cell lines after resveratrol administration. Materials and Methods: The experimental design included four cell culture groups: group 1: HL-60 (control), group 2: HL-60 + resveratrol, group 3: Raji, and group 4: Raji + resveratrol. Then, two repeated microarray analyses were performed. The different miRNA expressions were identified by bioinformatic analysis and after that, validation was performed by simultaneous polymerase chain reaction. Besides, apoptosis ratio was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical markers. Results: By microarray analysis, eight miRNAs (hsa-mir18 to, hsa-let-7d, hsa-let- 7b, hsa-mir-1246, hsa-miR-320b, hsa-miR-92a, hsa-miR-609 and hsa-miR-337-3p) which have different expression levels between the HL-60 (control) and HL60 + resveratrol groups were detected. There was one miRNA (hsa-miR-378) which was differently expressed between Raji (control) and Raji + resveratrol groups. Four miRNAs (miR-18a, miR-92a, miR-320a, miR-378) expression levels were confirmed with simultaneous polymerase chain reaction for validation. The apoptosis (TUNEL) rate of HL-60 + resveratrol group was two times higher compared to the HL-60 control group. There was no significant difference in apoptosis (TUNEL) rate between the groups of Raji control and Raji + resveratrol. However, it showed a tendency to increase in resveratrol-administrated Raji cells. However, in the immunohistochemical analysis, especially Fas and caspase markers were more prominently expressed. Conclusion: In this study; effects of resveratrol, natural bioactive substances, on HL-60 and Raji cells were evaluated and different miRNA expression levels were detected. In addition, cell death was thought to be especially through the caspase and Fas mechanism. However, considering their therapeutic potential in leukemia and lymphoma further functional and organism studies are needed.

Keywords

Resveratrol, microRNA, apoptosis, leukemia, lymphoma

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