Kemal ERGİN, Gökay BOZKURT, Cankut ÇUBUK, SAFİYE AKTAŞ

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

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.

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

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.

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Tome-Carneiro J, Larrosa M, Gonzalez-Sarrias A, Tomas-Barberan FA, Garcia-Conesa MT, Espin JC. Resveratrol and clinical trials: the crossroad from in vitro studies to human evidence. Curr Pharm Des 2013; 19: 6064-93.
Smoliga JM, Baur JA, Hausenblas HA. Resveratrol and health--a comprehensive review of human clinical trials. Mol Nutr Food Res 2011; 55: 1129-41.
Halliwell B. Dietary polyphenols: good, bad, or indifferent for your health? Cardiovasc Res 2007; 73: 341-7.
Bishayee A. Cancer prevention and treatment with resveratrol: From rodent studies to clinical trials. Cancer Prev Res 2009; 2: 409-18.
Alkhalaf M. Resveratrol-induced growth inhibition in MDA- MB-231 breast cancer cells is associated with mitogen-activated protein kinase signaling and protein translation. Eur J Cancer Prev 2007; 16: 334-41.
Fulda S, Debatin KM. Sensitization for tumor necrosis factor- related apoptosis-inducing ligand-induced apoptosis by thechemopreventive agent resveratrol. Cancer Res 2004; 64: 337-46.
Fukui M, Yamabe N, Kang KS, Zhu BT. Growth-stimulatory effect of resveratrol in human cancer cells. Mol Carcinog 2010; 49: 750-9.
Athar M, Back JH, Tang X, Kim KH, Kopelovich L, Bickers DR, et al. Resveratrol: a review of preclinical studies for human cancer prevention. Toxicol Appl Pharmacol 2007; 224: 274-83.
Stervbo U, Vang O, Bonnesen C. Time- and concentration- dependent effects of resveratrol in HL-60 and HepG2 cells. Cell Prolif 2006; 39: 479-93.
Kang JH, Park YH, Choi SW, Yang EK, Lee WJ. Resveratrol derivatives potently induce apoptosis in human promyelocytic leukemia cells. Exp Mol Med 2003; 35: 467-74.
Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ. Resveratrol, an antioxidant present in red wine, induces apoptosis in human promyelocytic leukemia (HL-60) cells. Cancer Lett 1999; 140: 1-10.
De Leo A, Arena G, Stecca C, Raciti M, Mattia E. Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt's lymphoma cells depending on viral latency program. Mol Cancer Res 2011; 9: 1346-55.
Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 2014; 15: 509-24.
Lee YS, Dutta A. MicroRNAs in cancer. Annu Rev Pathol 2009; 4: 199-227.
Randall G, Panis M, Cooper JD, Tellinghuisen TL, Sukhodolets KE, Pfeffer S, et al. Cellular cofactors affecting hepatitis C virus infection and replication. Proc Natl Acad Sci U S A 2007; 104: 12884-9.
Keller A, Leidinger P, Lange J, Borries A, Schroers H, Scheffler M, et al. Multiple sclerosis: microRNA expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls. PLoS One 2009; 4: e7440.
Löfgren SE, Frostegård J, Truedsson L, Pons-Estel BA, D'Alfonso S, Witte T, et al. Genes Immun. Genetic association of miRNA- 146a with systemic lupus erythematosus in Europeans through decreased expression of the gene. Genes Immun 2012; 13: 268-74.
Xiao B, Liu Z, Li BS, Tang B, Li W, Guo G, et al. Induction of microRNA-155 during Helicobacter pylori infection and its negative regulatory role in the inflammatory response. J Infect Dis 2009; 200: 916-25.
Nunez-Iglesias J, Liu CC, Morgan TE, Finch CE, Zhou XJ. Joint genome-wide profiling of miRNA and mRNA expression in Alzheimer's disease cortex reveals altered miRNA regulation. PLoS One 2010; 5: e8898.
Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005; 65: 7065-70.
Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006; 9: 189-98.
Volinia S, Galasso M, Sana ME, Wise TF, Palatini J, Huebner K, et al. Breast cancer signatures for invasiveness and prognosis defined by deep sequencing of microRNA. Proc Natl Acad Sci U S A 2012; 109: 3024-9.
Jansson MD, Lund AH. MicroRNA and cancer. Mol Oncol 2012; 6: 590-610.
Nelson KM, Weiss GJ. MicroRNAs and cancer: past, present, and potential future. Mol Cancer Ther 2008; 7: 3655-60.
Phuah NH, Nagoor NH. Regulation of microRNAs by natural agents: new strategies in cancer therapies. Biomed Res Int 2014; 2014: 804510.
Tili E, Michaille JJ. Resveratrol, MicroRNAs, Inflammation, and Cancer. J Nucleic Acids 2011; 2011: 102431.
Tili E, Michaille JJ, Alder H, Volinia S, Delmas D, Latruffe N, et al. Resveratrol modulates the levels of microRNAs targeting genes encoding tumor-suppressors and effectors of TGF? signaling pathway in SW480 cells. Biochem Pharmacol 2010; 80: 2057-65.
Dhar S, Hicks C, Levenson AS. Resveratrol and prostate cancer: promising role for microRNAs. Mol Nutr Food Res 2011; 55: 1219-29.
Liang H, Xia Q, Li P, Kong H, Lei P, Wang S, et al. Resveratrol induces apoptosis of pancreatic cancers cells by inhibiting miR-21 regulation of BCL-2 expression. Clin Transl Oncol 2013; 15: 741-6.
Boyerinas B, Park SM, Hau A, Murmann AE, Peter ME. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer 2010; 17: F19-36.
Li Z, Lu J, Sun M, Mi S, Zhang H, Luo RT, et al. Distinct microRNA expression profiles in acute myeloid leukemia with common translocations. Proc Natl Acad Sci 2008; 105:15535-40.
Garzon R, Volinia S, Liu CG, Fernandez-Cymering C, Palumbo T, Pichiorri F, et al. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood 2008; 111: 3183-9.
Ohyashiki JH, Umezu T, Kobayashi C, Hamamura RS, Tanaka M, Kuroda M, et al. 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.
Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 2005; 65: 9628-32.
Takakura S, Mitsutake N, Nakashima M, Namba H, Saenko VA, Rogounovitch TI, et al. Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells. Cancer Sci 2008; 99: 1147-54.
Haug BH, Henriksen JR, Buechner J, Geerts D, Tomte E, Kogner P, et al. MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma. Carcinogenesis 2011; 32: 1005-1012.
Sharifi M, Salehi R, Gheisari Y, Kazemi M. Inhibition of microRNA miR-92a induces apoptosis and inhibits cell proliferation in human acute promyelocytic leukemia through modulation of p63 expression. Mol Biol Rep 2014; 41: 2799-808.