The Regulatory Role and Therapeutic Effect of miR-196a on Autophagy in Pancreatic Ductal Adenocarcinoma Cells

Öz

Objective: Pancreatic cancer is one of the most aggressive and lethal cancers due to its late diagnosis and high metastatic potential. Although autophagy is defined as a cell death mechanism under physiological conditions, it is a required survival mechanism to continue cancer proliferation. Therefore, autophagy must be inhibited for the treatment of cancer in the metastatic stage. This can only be achieved by defining top mediators that play a role in the control of autophagy. In our study, it was aimed to investigate the role of miR-196a, which has an oncogenic effect, and its suppression in its follow-up, the possible therapeutic effects on pancreatic cancer cells through inhibition of autophagy. Material-Method: Followed by transfection of Panc-1 and MiaPaCa-2 cells with control-miR or miR-196a inhibitor, the cell viability and mRNA / protein expressions of Beclin-1, Atg5 and Atg12, which are important mediators of cell viability and autophagy, were evaluated by MTS, RT-PCR and western blot methods, respetively. Results: We showed that miR-196a is related to the viability of PDAC cells and also miR-196a inhibition significantly reduced PDAC cells proliferation without any cellular toxic effects. It was also shown that miR-196a inhibitor transfection significantly inhibited Beclin-1 and Atg5 gene expressions, especially in Panc-1, whereas increased Atg5 and Atg12 protein expressions in MiaPaCa-2. In Panc-1 and MiaPaCa-2 cells, miR-196a treatment did not have a significant effect on Beclin-1 protein expression and did not cause a difference between cells. Atg5 protein expression was higher in MiaPaCa-2 cells. Conclusion: miR-196 inhibition was shown to decrease autophagy in metastatic Panc-1 cells and induce autophagy in MiaPaCa-2, whose metastatic properties are not defined. Therefore, miR-196a inhibitor therapeutic approach decreases the proliferation of PDAC cells through regulating the autophagy that is impaired in cancer.

Anahtar Kelimeler

• miRNA
• miR196a
• pancreatic cancer
• autophagy

Pankreatik Duktal Adenokarsinoma Hücrelerinde miR-196a’nın, Otofajinin Kontrolü Üzerindeki Rolü ve Terapötik Etkinliği

Öz

Amaç: Pankreas kanseri, geç dönemde tanı konulması ve yüksek metastatik potansiyele sahip olması nedeniyle en agrasif ve en ölümcül kanserlerin başında yer almaktadır. Fizyolojik şartlarda bir hücre ölüm mekanizması olarak tanımlanan otofaji, aslında tersine kanserde canlılığın devamı için gerekli major mekanizmadır. Bu nedenle, metastatik aşamadaki kanserin tedavisi için otofaji inhibe edilmelidir. Bu da ancak otofajinin kontrolünde rol oynayan üst mediyatörlerin tanımlanması ile sağlanabilecektir. Çalışmamızda, onkojenik etkiye sahip olan miR-196a’nın rolünün ve takibinde baskılanmasının, otofajinin inhibisyonu üzerinden pankreas kanseri hücrelerinde olası terapötik etkilerinin incelenmesi amaçlanmıştır. Materyal-Metot: Pankreatik duktal adenokarsinoma (PDAC) hücreleri olan Panc-1 ve MiaPaCa-2’nin negatif-miR veya miR-196a inhibitör ile transfeksiyonu sonrasında; hücre canlılığı ve otofajinin önemli mediyatörlerinden olan Beklin-1, Atg5 ve Atg12’nin mRNA ve protein ekspresyonları sırasıyla MTS, RT-PCR ve western blot yöntemleri ile değerlendirilmiştir. Bulgular: Sonuçlarımız; miR-196a’nın, PDAC hücrelerinin canlılığı ile ilişkili olduğunu ve miR-196a inhibisyonunun herhangi bir hücresel toksik etki oluşturmadan PDAC hücrelerinin canlılığını anlamlı düzeyde azalttığını göstermiştir. Ayrıca miR-196a inhibitör uygulamasının, özellikle Panc-1’da Beklin-1 ve Atg5 gen ekspresyonunu önemli düzeyde inhibe ettiğini, ayrıca tersine MiaPaCa-2’de Atg5 ve Atg12 protein ekspresyonunu arttırdığını göstermiştir. Panc-1 ve MiaPaCa-2 hücrelerinde miR-196a uygulamaları Beklin-1 protein ekspresyonları üzerinde önemli bir etki yaratmamış ve hücreler arasında bir farklılığa yol açmamıştır. Atg5 protein ekspresyonu ise MiaPaCa-2 hücrelerinde daha yüksek bulunmuştur. Sonuç: miR-196 inhibisyonunun, ileri evre Panc-1 hücresinde otofajiyi inhibe, metastatik özellikleri tanımlanmamış olan MiaPaCa-2’de ise otofajiyi uyardığını saptamış bulunuyoruz. Dolayısıyla miR-196a inhibitör tedavisi yaklaşımı, kanserde kontrolü bozulan otofajiyi düzenleyerek PDAC hücrelerinin proliferasyonu azaltmaktadır.

Anahtar Kelimeler

• miRNA
• miR196a
• pankreas kanseri
• otofaji

Kaynakça

1. 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. Cancer J Clin. 2012;62:10-29.
2. 2. Decker GA, Batheja MJ, Collins JM, Silva AC, Mekeel KL, Moss AA, et al. Risk factors for pancreatic adenocarcinoma and prospects for screening. Gastroentol Hepatol. 2010;6(4):246.
3. 3. Lowenfels AB, Maisonneuve P. Epidemiology and risk factors for pancreatic cancer. Best Pract Res Clin Gastroenterol. 2006;20(2):197-209.
4. 4. Stolzenberg-Solomon RZ, Blaser MJ, Limburg PJ, Perez-Perez G, Taylor PR, Virtamo J, et al. Helicobacter pylori seropositivity as a risk factor for pancreatic cancer. J Natl Cancer Inst. 2001;93(12):937-41.
5. 5. Korc M, Jeon CY, Edderkaoui M, Pandol SJ, Petrov MS. Tobacco and alcohol as risk factors for pancreatic cancer. Best Pract Res Clin Gastroenterol. 2017;31(5):529-36.
6. 6. Eibl G, Cruz-Monserrate Z, Korc M, Petrov MS, Goodarzi MO, Fisher WE, et al. Diabetes Mellitus and Obesity as Risk Factors for Pancreatic Cancer. J Acad Nutr Diet. 2018;118(4):555-67.
7. 7. Kocaturk NM, Akkoc Y, Kig C, Bayraktar O, Gozuacik D, Kutlu O. Autophagy as a molecular target for cancer treatment. Eur J Pharm Sci. 2019;15(134):116-37.
8. 8. Albulescu R, Popa AC, Codrici E, Popescu DI, Mihai S, Tanase C. (Editor: Kelly McCall) Pancreatic Cancer - Insights into Molecular Mechanisms and Novel Approaches to Early Detection and Treatment. Chapter: 3: miRNAs in Pancreatic Cancer. 2014; DOI: 10.5772/58397.

9. 9. Yanokura M, Banno K, Iida M, Irie H, Umene K, Masuda K, et al. MicroRNAs in Endometrial Cancer: Recent Advances and Potential Clinical Applications. EXCLI J. 2015;2(14):190-8.
10. 10. Gurbuz N, Ozpolat B. MicroRNA-based Targeted Therapeutics in Pancreatic Cancer. Anticancer Res. 2019;39:529-32.
11. 11. Fantini S, Salsi V, Vitobello A, Rijli FM, Zappavigna V. MicroRNA-196b is transcribed from an autonomous promoter and is directly regulated by Cdx2 and by posterior Hox proteins during embryogenesis. Biochim Biophys Acta. 2015;1849(8):1066-80.
12. 12. Szafranska AE, Doleshal M, Edmunds HS, Gordon S, Luttges J, Munding JB, et al. Analysis of microRNAs in pancreatic fine-needle aspirates can classify benign and malignant tissues. Clin Chem. 2008;54(10):1716-24.
13. 13. Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
14. 14. Lu YC, Chang JT, Chan EC, Chao YK, Yeh TS, Chen JS, et al. miR-196, an Emerging Cancer Biomarker for Digestive Tract Cancers. J Cancer. 2016;7(6): 650-5.
15. 15. Chen C, Zhang Y, Zhang L, Weakley SM, Yao Q. MicroRNA-196: Critical Roles and Clinical Applications in Development and Cancer. J Cell Mol Med. 2011;15(1):14-23.
16. 16. Laurila EM, Sandstro S, Rantanen LM, Autio R, Kallioniemi A. Both Inhibition and Enhanced Expression of miR-31 Lead to Reduced Migration and Invasion of Pancreatic Cancer Cells. Genes Chromosom Cancer. 2012;51(6):557-68.
17. 17. Lee YS, Kim H, Kim HW, Lee JC, Paik KH, Kang J, et al. High Expression of MicroRNA-196a Indicates Poor Prognosis in Resected Pancreatic Neuroendocrine Tumor. Medicine. 2015;94(50):1-8.
18. 18. Xue Y, Tayoun ANA, Abo KM, Pipas JM, Gordon SR, Gardner TB, Bart Jr RJ, Suriawinata AA, Tsongalis GJ. MicroRNAs as diagnostic markers for pancreatic ductal adenocarcinoma and its precursor, pancreatic intraepithelial neoplasm. Cancer Genetics. 2013;206(6):217-21.
19. 19. Liu-Minghao BS, Du- Yiqi MD, Gao- Jun MD, Liu- Jianqiang MD, Kong- Xiangyu MD, Gong- Yanfang BS, et al. Aberrant Expression miR-196a is Associated With Abnormal Apoptosis, Invasion, and Proliferation of Pancreatic Cancer Cells. Pancreas. 2013;42(7):1169-81.
20. 20. Huang F, Tang J, Zhuang X, Zhuang Y, Cheng W, Chen W, Yao H, Zhang S. MiR-196a Promotes Pancreatic Cancer Progression by Targeting Nuclear Factor Kappa-B-Inhibitor Alpha. PLoS One. 2014;9(2):1-9.
21. 21. Liu B, Wen X, Cheng Y. Survival or death: disequilibrating the oncogenic and tumor suppressive autophagy in cancer. Cell Death Dis. 2013;4(10):e892.
22. 22. Song YJ, Zhang SS, Guo XL, Sun K, Han ZP, Li R, et al. Autophagy contributes to the survival of CD133+ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment. Cancer Lett. 2013;339(1):70-81.
23. 23. Zhu H, Wu H, Liu X, Li B, ChenY, Ren X, et al. Research Paper Regulation of autophagy by a Beclin 1-targeted microRNA, miR-30a, in cancer cells. Autophagy. 2009;5(6):816-23.