The Role of Synchronization Mediators in Circadian Rhythm in HT-29 Colon Cancer Cell Line

Abstract

In mammals, the central circadian clock is located in the hypothalamus, coordinating peripheral clocks with neuronal and endocrine signals. It has been shown that circadian clock genes and proteins continue their rhythmic expression in peripheral cells and tissues independent of central clock. The rhythm continues even in the immortalized cell lines, but intercellular coordination is impaired. Cell lines are good mediators for studying cancer-related pathways and circadian rhythm relationships. However, it should first be shown that rhythmic gene expression continues. Some mediators are used to achieve intercellular synchronization. The aim of this study was to investigate the role of strong synchronization initiators, FBS, and dexamethasone, on the expression of the PER2 clock gene in the HT-29 colon cancer cell line. For this purpose; following FBS or dexamethasone administration, HT-29 cells were harvested at 6 different times to isolate RNA and the PER2 and ACTB gene expressions were quantified by real-time PCR assay. The results were evaluated by Linear Cosinor analysis. When the change in mRNA expression of the PER2 gene was compared to the ACTB reference gene, it was shown that rhythmic expression was achieved with both initiators. According to the Cosinor analysis, a significant rhythmic change was observed in both 18 and 24 hour periods. However, in the synchronization with dexamethasone, the confidence interval in the time Period was also significant in the 24 hour period. In this study, it was determined that the initiators may vary in the calculation of circadian Periods which was carried out with two different initiators in HT-29 cell line.

Keywords

• Synchronization,Circadian rthym,HT-29,FBS,Dexamethasone

HT-29 Kolon Kanser Hücre Hattında Senkronizasyon Başlatıcıların Sirkadiyen Ritimdeki Rolü

Abstract

Memelilerde merkezi sirkadiyen saat, hipotalamusta konumlanmıştır. Periferal saatleri nöronal ve endokrin sinyaller ile koordine etmektedir. Sirkadiyen saat gen ve proteinlerinin, merkezi saatten bağımsız olarak da periferal hücre ve dokularda ritmik ekspresyona devam ettiği yapılan çalışmalar ile gösterilmiştir. İmmortalize hücre hatlarında da ritim devam etmekte ancak hücreler arası koordinasyon bozulmaktadır. Hücre hatları kanser ile ilgili yolaklar ve sirkadiyen ritim ilişkisini çalışmak için en elverişli aracılardır. Ancak öncelikle ritmik gen ekspresyonunun devam ettiğinin gösterilmesi gerekmektedir. Hücreler arası senkronizasyonun sağlanması için bazı aracılar kullanılmaktadır. Bu çalışmadaki amacımız; güçlü senkronizasyon başlatıcılar olan fetal bovin serum ve deksametazonun HT-29 kolon kanseri hücre hattında PER2 saat geninin ekspresyonu üzerindeki rolünü incelemektir. Bu amaçla; fetal bovin serum veya deksametazon uygulamasını takiben HT-29 hücreleri altı farklı zamanda toplanarak RNA izole edilmiş ve PER2 ve ACTB gen ekspresyonları gerçek zamanlı PZR deneyi ile kantifiye edilmiştir. Sonuçlar Lineer Cosinor analizleri ile değerlendirilmiştir. PER2 genine ait mRNA ekspresyonlarındaki değişim ACTB referans geni ile kıyaslanarak değerlendirildiğinde, her iki başlatıcı ile de ritmik ekspresyonun sağlandığı gösterilmiştir. Her iki başlatıcı ile de Cosinor analizine göre 18 ve 24 saatlik periyot sürelerinde anlamlı ritmik değişim gösterilmiştir. Ek olarak deksametazon ile başlatılan senkronizasyonda, 24 saatlik periyot süresinde zaman dilimindeki güven aralığının anlamlı olduğu gözlenmiştir. HT-29 hücre hattında iki farklı başlatıcı ile yapılan bu çalışmada sirkadiyen periyot sürelerinin hesaplanmasında başlatıcıların değişkenlik gösterebildiği tespit edilmiştir.

Keywords

• Senkronizasyon,Sirkadiyen ritim,HT-29,FBS,Deksametazon

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