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

Abstract

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.

Keywords

• miRNA
• miR196a
• pancreatic cancer
• autophagy

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

Abstract

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.

Keywords

• miRNA
• miR196a
• pankreas kanseri
• otofaji

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