The Role of Reference Gene Differences in Synchronization Control of Circadian Rhythm-Induced Gene Expression in the HT29 Cell Line

Öz

Circadian timing system controls many important vital functions through molecular clocks in mammalian organs and cells. The role of circadian rhythm in diseases has been better understood in recent years. In cell culture studies to be carried out in this area, primarily important things are ensuring synchronization and its control. In this study, the synchronization was controlled by PER2 clock gene and the two reference genes in nutrient-mediated synchronized HT-29 cells. Our aim is to investigate the role of reference genes in synchronization control. For this purpose, following serum shock HT-29 cells were harvested at 6 different times, RNA isolation was made and  PER2, ACTB, and RPLP0 gene expressions were quantified by real-time PCR (RT-PCR) assay. The results were evaluated with Fourier transformation and Linear Cosinor analysis. When the ACTB reference gene was selected, 24 and 30 hour periods showed a significant change by Fourier analysis and 24-hour period showed a significant change with Cosinor analysis. When the RPLP0 reference gene was selected, the 18 hour cycle time was significant with both analyzes. As the findings show, it is important to evaluate several parameters such as reference gene and analysis method in the initiation and control of synchronization. It is thought that this study will create awareness for in vitro studies in this area.

Anahtar Kelimeler

• Synchronization,Circadien rthym,HT29,PER2

HT29 Hücre Hattında Sirkadiyen Ritme Bağlı Gen İfadesinin Kontrolünde Referans Gen Farklılığının Senkronizasyondaki Rolü

Öz

Sirkadiyen zamanlama sistemi, memeli organ ve hücrelerinde bulunan moleküler saatler aracılığı ile pek çok hayati fonksiyonu kontrol etmektedir. Son yıllarda yapılan çalışmalar ile sirkadiyen ritmin hastalıklardaki rolü daha iyi anlaşılmıştır. Bu kapsamda gerçekleştirilecek hücre kültürü çalışmalarında öncelikli olarak senkronizasyonun sağlanması ve kontrolü önem arz etmektedir. Sunduğumuz çalışmada besin aracılığı ile senkronize edilen HT-29 hücrelerinde, senkronizasyon saat geni olarak da bilinen PER2 ve iki farklı referans gen ile kontrol edilmiştir. Amacımız senkronizasyon kontrolünde referans genlerin rolünü incelemektir. Bu amaçla; serum şokunu takiben HT-29 hücreleri T0 zamanından itibaren 6 farklı zamanda toplanarak RNA izole edilmiş ve PER2, ACTB ve RPLP0 gen ekspresyonları gerçek zamanlı PZR (RT-PZR) deneyi ile kantifiye edilmiştir. Sonuçlar Fourier transformasyon ve Lineer Cosinor analizleri ile değerlendirilmiştir. ACTB referans geni seçildiğinde Fourier transformasyon analizi ile 24 ve 30 saatlik periyotlar anlamlı değişim gösterirken, Lineer Cosinor analizi ile sadece 24 saatlik periyot süresi anlamlı değişim göstermiştir. RPLP0 referans geni seçildiğinde her iki analiz ile de 18 saatlik periyot süresi anlamlılık göstermiştir. Bulguların gösterdiği gibi senkronizasyonun başlatılması ve kontrolünde referans gen ve analiz yöntemi gibi pek çok parametrenin birlikte değerlendirilmesi önem arz etmektedir. Yapılan çalışmanın bu alanda yapılacak in vitro çalışmalar için farkındalık yaratacağı düşünülmektedir. 

Anahtar Kelimeler

• Senkronizasyon,Sirkadiyen ritim,HT29,PER2

Kaynakça

1. Akashi, M., & Takumi, T. (2005). The orphan nuclear receptor RORalpha regulates circadian transcription of the mammalian core-clock Bmal1. Nat Struct Mol Biol, 12(5), 441-448. doi:10.1038/nsmb925
2. Bairoch, A. (2018). The Cellosaurus, a Cell-Line Knowledge Resource. J Biomol Tech, 29(2), 25-38. doi:10.7171/jbt.18-2902-002
3. Balsalobre, A., Damiola, F., & Schibler, U. (1998). A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell, 93(6), 929-937.
4. Brody, H. (2015). Colorectal cancer. Nature, 521(7551), S1. doi:10.1038/521S1a
5. Cornelissen, G. (2014). Cosinor-based rhythmometry. Theor Biol Med Model, 11, 16. doi:10.1186/1742-4682-11-16
6. Dallmann, R., Okyar, A., & Levi, F. (2016). Dosing-Time Makes the Poison: Circadian Regulation and Pharmacotherapy. Trends Mol Med, 22(5), 430-445. doi:10.1016/j.molmed.2016.03.004
7. Debruyne, J. P., Noton, E., Lambert, C. M., Maywood, E. S., Weaver, D. R., & Reppert, S. M. (2006). A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron, 50(3), 465-477. doi:10.1016/j.neuron.2006.03.041
8. Fourier, J. (1822). Théorie Analytique de la Chaleur. Paris, France.

9. Izumo, M., Sato, T. R., Straume, M., & Johnson, C. H. (2006). Quantitative analyses of circadian gene expression in mammalian cell cultures. PLoS Comput Biol, 2(10), e136. doi:10.1371/journal.pcbi.0020136
10. Katamune, C., Koyanagi, S., Hashikawa, K. I., Kusunose, N., Akamine, T., Matsunaga, N., & Ohdo, S. (2018). Mutation of the gene encoding the circadian clock component PERIOD2 in oncogenic cells confers chemoresistance by up-regulating the Aldh3a1 gene. J Biol Chem. doi:10.1074/jbc.RA118.004942
11. Lesicka, M., Jablonska, E., Wieczorek, E., Seroczynska, B., Siekierzycka, A., Skokowski, J., Reszka, E. (2018). Altered circadian genes expression in breast cancer tissue according to the clinical characteristics. PLoS One, 13(6), e0199622. doi:10.1371/journal.pone.0199622
12. Levi, F., Okyar, A., Dulong, S., Innominato, P. F., & Clairambault, J. (2010). Circadian timing in cancer treatments. Annu Rev Pharmacol Toxicol, 50, 377-421. doi:10.1146/annurev.pharmtox.48.113006.094626
13. Marquardt, D. W. (1963). An Algorithm for Least-Squares Estimation of Nonlinear Parameters. Journal of the Society for Industrial and Applied Mathematics, 11(2), 431–441. doi:https://doi.org/10.1137/0111030
14. Mazzoccoli, G., Vinciguerra, M., Papa, G., & Piepoli, A. (2014). Circadian clock circuitry in colorectal cancer. World J Gastroenterol, 20(15), 4197-4207. doi:10.3748/wjg.v20.i15.4197
15. Minami, Y., Ode, K. L., & Ueda, H. R. (2013). Mammalian circadian clock: the roles of transcriptional repression and delay. Handb Exp Pharmacol(217), 359-377. doi:10.1007/978-3-642-25950-0_15
16. Momma, T., Okayama, H., Saitou, M., Sugeno, H., Yoshimoto, N., Takebayashi, Y., Takenoshita, S. (2017). Expression of circadian clock genes in human colorectal adenoma and carcinoma. Oncol Lett, 14(5), 5319-5325. doi:10.3892/ol.2017.6876
17. Mteyrek, A., Filipski, E., Guettier, C., Okyar, A., & Levi, F. (2016). Clock gene Per2 as a controller of liver carcinogenesis. Oncotarget, 7(52), 85832-85847. doi:10.18632/oncotarget.11037
18. Nelson, W., Tong, Y. L., Lee, J. K., & Halberg, F. (1979). Methods for cosinor-rhythmometry. Chronobiologia, 6(4), 305-323.
19. Ozturk, N., Ozturk, D., Kavakli, I. H., & Okyar, A. (2017). Molecular Aspects of Circadian Pharmacology and Relevance for Cancer Chronotherapy. Int J Mol Sci, 18(10). doi:10.3390/ijms18102168.
20. Özbayer, C. (2011). Circadian clock, cell cycle, and cancer. Dicle Medical Journal / Dicle tıp Dergisi, 38(4), 514-518. doi:10.5798/diclemedj.0921.2011.04.0080Pardini, L., Kaeffer, B., Trubuil, A., Bourreille, A., & Galmiche, J. P. (2005). Human intestinal circadian clock: expression of clock genes in colonocytes lining the crypt. Chronobiol Int, 22(6), 951-961. doi:10.1080/07420520500395011.
21. Pilanci, K. N., Saglam, S., Okyar, A., Yucel, S., Pala-Kara, Z., Ordu, C., Kaytan-Saglam, E. (2016). Chronomodulated oxaliplatin plus Capecitabine (XELOX) as a first line chemotherapy in metastatic colorectal cancer: A Phase II Brunch regimen study. Cancer Chemother Pharmacol, 78(1), 143-150. doi:10.1007/s00280-016-3067-x.
22. Puram, R. V., Kowalczyk, M. S., de Boer, C. G., Schneider, R. K., Miller, P. G., McConkey, M., Ebert, B. L. (2016). Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML. Cell, 165(2), 303-316. doi:10.1016/j.cell.2016.03.015.
23. Refinetti, R., Lissen, G. C., & Halberg, F. (2007). Procedures for numerical analysis of circadian rhythms. Biol Rhythm Res, 38(4), 275-325. doi:10.1080/09291010600903692.
24. Reszka, E., & Zienolddiny, S. (2018). Epigenetic Basis of Circadian Rhythm Disruption in Cancer. Methods Mol Biol, 1856, 173-201. doi:10.1007/978-1-4939-8751-1_10.
25. Sato, T. K., Panda, S., Miraglia, L. J., Reyes, T. M., Rudic, R. D., McNamara, P., Hogenesch, J. B. (2004). A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron, 43(4), 527-537. doi:10.1016/j.neuron.2004.07.018
26. Schibler, U. (2005). The daily rhythms of genes, cells, and organs. Biological clocks and circadian timing in cells. EMBO Rep, 6 Spec No, S9-13. doi:10.1038/sj.embor.7400424
27. Schibler, U., Gotic, I., Saini, C., Gos, P., Curie, T., Emmenegger, Y., Franken, P. (2015). Clock-Talk: Interactions between Central and Peripheral Circadian Oscillators in Mammals. Cold Spring Harb Symp Quant Biol, 80, 223-232. doi:10.1101/sqb.2015.80.027490
28. Siegel, R. L., Miller, K. D., Fedewa, S. A., Ahnen, D. J., Meester, R. G. S., Barzi, A., & Jemal, A. (2017). Colorectal cancer statistics, 2017. CA Cancer J Clin, 67(3), 177-193. doi:10.3322/caac.21395.
29. Türkiye Kanser İstatistikleri. (2015).
30. Winter, S. L., Bosnoyan-Collins, L., Pinnaduwage, D., & Andrulis, I. L. (2007). Expression of the circadian clock genes Per1 and Per2 in sporadic and familial breast tumors. Neoplasia, 9(10), 797-800.
31. Wood, P. A., Yang, X., & Hrushesky, W. J. (2009). Clock genes and cancer. Integr Cancer Ther, 8(4), 303-308. doi:10.1177/1534735409355292Yang, M. Y., Yang, W. C., Lin, P. M., Hsu, J. F., Hsiao, H. H., Liu, Y. C., Lin, S. F. (2011). Altered expression of circadian clock genes in human chronic myeloid leukemia. J Biol Rhythms, 26(2), 136-148. doi:10.1177/0748730410395527.
32. Zhang, J., Dong, X., Fujimoto, Y., & Okamura, H. (2004). Molecular signals of Mammalian circadian clock. Kobe J Med Sci, 50(3-4), 101-109.