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

Hücre Serilerinde Kilitli Nükleik Asitler İle MIR223 Gen Sessizleştirmesi

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

References

1. 1. Lee RC, Feinbaum RL and Ambros V. The c. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993;75:843-54.
2. 2. Mochizuki K, Fine NA, Fujisawa T, Gorovsky MA. Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena. Cell 2002;110:689-99.
3. 3. Peragine A, Yoshikawa M, Wu G, Albrecht HL, Poethig RS. SG3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans acting siRNAs in Arabidopsis. Genes Dev 2004;18:2368-79.
4. 4. O’Donnell KA and Boeke JD. Mighty Piwis defend the germline against genome intruders. Cell 2007;129:37-44.
5. 5. Vishnoi A, Rani S. MiRNA Biogenesis and Regulation of Diseases: An Overview. Methods Mol Biol 2017;1509:1-10.
6. 6. Sun W, Shen W, Yang S, Hu F, Li H, Zhu TH. MIR223 and miR-142 attenuate hematopoietic cell proliferation, and MIR223 positively regulates miR-142 through LMO2 isoforms and CEBP-β. Cell Res 2010;20(10):1158–69.
7. 7. Fulci V, Scappucci G, Sebastiani GD, Giannitti C, Franceschini D, Meloni F. et. al. MIR223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol 2010;71(2):206-11.
8. 8. Han L, Rachel B, Stuart C. MicroRNA-223 regulates Glut4 expression and cardiomyocyte glucose metabolism. Cardiovasc Res 2010;86(3):410-20.

9. 9. Haneklaus M, Gerlic M, O’Neill LAJ, Masters SL. MIR223: infection, inflammation and cancer. J Intern Med 2013;274(3);215-26.
10. 10. Mansour MR, Sanda T, Lawton LN, Li X, Kreslavsky T, Novina CD, Brand M et.al. The TAL1 complex targets the FBXW7 tumor suppressor by activating MIR223 in human T cell acute lymphoblastic leukemia. J Exp Med 2013;210(8):1545-57. 
11. 11. Yimei C, Xiaomin Y, Songnian H, Jun Yu. A brief review on the mechanisms of miRNA regulation. Genomics Proteomics Bioinformatics 2009;7(4):147-54.
12. 12. Mardani R, Abadi MHJN, Taghizadeh-Boroujeni S, Bayat A, Farsinezhad A, Hayat SMG. et. al. MicroRNA in Leukemia: Tumor Suppressors and Oncogenes with Prognostic Potential. J Cell Physiol 2018;234(6):8465-86.
13. 13. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E. et.al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002;99(24):15524-29.
14. 14. Tanaka M, Oikawa K, Takanashi M, Kudo M, Ohyashiki J, Ohyashiki K. et.al. Downregulation of miR-92 in human plasma is a novel marker for acute leukemia patients. PLoS One 2009;4(5):e5532.
15. 15. Ohyashiki JH, Umezu T, Kobayashi C, Hamamura RS, Tanaka M, Kuroda M. Impact on cell to plasma ratio of miR-92a in patients with acute leukemia: in vivo assessment of cell to plasma ratio of miR-92a. BMC Res Notes 2010;3:347.
16. 16. Den Boer ML, van Slegtenhorst M, De Menezes RX, Cheok MH, Buijs-Gladdines JG, Peters ST et. al. A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome-wide classification study. Lancet Oncol 2009;10(2):125-34.
17. 17. Schotte D, Akbari-Moqadam F, Lange-Turenhout EA, Chen C, Pieters R, Den Boer ML. Discovery of new microRNAs by small RNAome deep sequencing in childhood acute lymphoblastic leukemia. Leukemia 2011;25(9):1389-99.
18. 18. Bellon M, Yves L, Hermine O. and Nicot C. Deregulation of micorRNA involved in hematopoiesis and the immune response in HTLV-I adult T-cell leukemia. Blood 2009;113(20):4914-17.
19. 19. Chen C, Li L, Lodish HF. MicroRNAs modulate hematopoietic lineage differentation. Science 2004;303(5654):83-6.
20. 20. Stenvang J, Silahtaroglu AN, Lindow M, Elmen J, Kauppinen S. The utility of KNA in microRNA based cancer diagnostics and therapeutics. Semin Cancer Biol 2008;18(2):89-102.
21. 21. Grünweller A, Hartmann RK. Locked nucleic acid oligonucleotides: the next generation of antisense agents? BioDrugs 2007;21(4):235-43. 
22. 22. Nedaeinia R, Avan A, Ahmadian M, Nia SN, Ranjbar M, Sharifi M. et al. Piroozmand A, Nourmohammadi E, Manian M, Ferns GA, Ghayour-Mobarhan M, Salehi R. Current Status and Perspectives Regarding KNA-AntimiR Oligonucleotides and microRNA miR-21 Inhibitors as a Potential Therapeutic Option in Treatment of Colorectal Cancer. J Cell Biochem 2017;118(12):4129-40. 
23. 23. Landgraf, P., M. Rusu, R. Sheridan, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007;129(7):1401-14.