Gamze CÖMERTPAY, Ümit LÜLEYAP, Mehmet Bertan YILMAZ, Turan TUFAN, Perçin PAZARCI

İnsan amniotik hücre kültüründe nikotin ve resveratrolün Sox2 ve Sox4 genlerinin ekspresyon seviyeleri üzerindeki etkilerinin real time PCR ile araştırılması

Amaç: Sigarada bulunan en önemli toksik bileşenlerden bir tanesi nikotindir. Prenatal dönemde sigara dumanına maruz kalmak yeni doğanda bilişsel, duygusal, davranışsal problemler ve çocukluk kanserleriyle birlikte birçok farklı problem ortaya çıkarabilmektedir. Bu çalışmada, nikotinin master gen özelliklerine sahip genlerden olan SOX2 ve SOX4 genlerinin ekspresyon düzeylerine etkilerinin insan amniyotik hücre kültüründe araştırılması amaçlanmıştır. Ek olarak, bir antioksidan olan resveratrolün nikotin ile muamele edilmiş hücrelerde bu genlerin ekspresyon düzeylerine etkileri insan amniyotik hücre kültüründe incelenmiştir.Yöntem: Çalışmaya 20 hasta dahil edilmiştir ve her hasta için kontrol, nikotin ve nikotin+resveratrol hücre kültürü grupları oluşturulmuştur. SOX2 ve SOX4 genlerinin ekspresyon düzeyleri her grup için real time RT-PCR yöntemi kullanılarak tespit edilmiştir.Bulgular: SOX2 ve SOX4 genleri için nikotin ile muamele edilen grupta tespit edilen ekspresyon düzeylerindeki değişim istatistiksel olarak anlamlı bulunmuştur (p= 0.005 SOX2, p=0.000 SOX4). Ayrıca, nikotin ve nikotin+resveratrol ile muamele edilmiş gruplar karşılaştırıldığında sonuç istatistiksel olarak anlamlı bulunmuştur.Sonuç: Nikotin, insan amniyotik hücrelerinde SOX2 ve SOX4 genlerinin ekspresyon düzeylerini %60 oranında artırmıştır ve resveratrolün bu artmış ekspresyon düzeylerini düşüren önemli bir antioksidan olduğu tespit edilmiştir.

Anahtar Kelimeler:

Amniyotik Hücre Kültürü, Nikotin, Resveratrol, SOX2, SOX4

Investigation of the effects of nicotine and resveratrol on expression levels of Sox2 and Sox4 genes in human amniotic cell culture by real time PCR

Purpose: The aim of this study was to show the effects of nicotine on expression levels of SOX2 and SOX4 genes, which are known for their master gene features, in human amniotic cell cultures. The study also aimed to show the effects of resveratrol, an antioxidant, on expression levels of these genes in human amniotic cell cultures, which are treated by nicotine.Material and Methods: Twenty patients were included in this study and for each patient; control, nicotine treated and nicotine + resveratrol treated cell culture groups are formed. The expression levels of SOX2 and SOX4 genes are examined in each group by using real time RT-PCR.Results: Change in expression levels of SOX2 and SOX4 genes in nicotine treated group were found to be statistically significant. Also, when groups treated with nicotine and nicotine + resveratrol were compared the difference was found to be statistically significant.Conclusions: Nicotine increased the expression levels of SOX2 and SOX4 genes by 60% in human amniotic cell cultures and resveratrol was found to be an important antioxidant that reduces the increased expression levels of SOX2 and SOX4 genes caused by nicotine treatment.

Keywords:

Amniotic Cell Culture, Nicotine, Resveratrol, SOX2, SOX4,

PDF

___

1. Robinson DM, Peebles KC, Kwok H, Adams BM, Clarke LL, Woollard GA et al. Prenatal nicotine exposure increases apnoea and reduces nicotinic potentiation of hypoglossal inspiratory output in mice. J Physiol. 2002;538:957-73.
2. Bruchova H, Vasikova A, Merkerova M, Milcova A, Topinka J, Balascak I et al. Effect of maternal tobacco smoke exposure on the placental transcriptome. Placenta. 2010;31:186-91.
3. Rogers JM. Tobacco and pregnancy: overview of exposures and effects. Birth Defects Res C Embryo Today. 2008;84:1-15. 4. Wickstrom R. Effects of nicotine during pregnancy: human and experimental evidence. Curr Neuropharmacol. 2007;5:213-22.
5. Kirby PJ, Waters PD, Delbridge M, Svartman M, Stewart AN, Nagai K et al. Cloning and mapping of platypus SOX2 and SOX14: insights into SOX group B evolution. Cytogenet Genome Res. 2002;98:96-100.
6. Lin F, Lin P, Zhao D, Chen Y, Xiao L, Qin W et al. Sox2 targets cyclinE, p27 and survivin to regulate androgen-independent human prostate cancer cell proliferation and apoptosis. Cell Prolif. 2012;45:207-16.
7. Scharer CD, McCabe CD, Ali-Seyed M, Berger MF, Bulyk ML, Moreno CS. Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells. Cancer Res. 2009;69:709-17.
8. Hur W, Rhim H, Jung CK, Kim JD, Bae SH, Jang JW et al. SOX4 overexpression regulates the p53-mediated apoptosis in hepatocellular carcinoma: clinical implication and functional analysis in vitro. Carcinogenesis. 2010;31:1298-307.
9. Zhang H, Shih A, Rinna A, Forman HJ. Exacerbation of tobacco smoke mediated apoptosis by resveratrol: an unexpected consequence of its antioxidant action. Int J Biochem Cell Biol. 2011;43:1059-64.
10. Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS, Zhang C et al. Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations. Am J Physiol Heart Circ Physiol. 2008;294:H2721-35.
11. Hylkema MN, Blacquiere MJ. Intrauterine effects of maternal smoking on sensitization, asthma, and chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2009;6:660-2.
12. Jacob N, Golmard JL, Berlin I. Fetal exposure to tobacco: nicotine and cotinine concentration in amniotic fluid and maternal saliva. J Matern Fetal Neonatal Med. 2017;30:233-9.
13. Somm E, Schwitzgebel VM, Vauthay DM, Aubert ML, Huppi PS. Prenatal nicotine exposure and the programming of metabolic and cardiovascular disorders. Mol Cell Endocrinol. 2009;304:69-77.
14. Feng JH, Yan YE, Liang G, Liu YS, Li XJ, Zhang BJ et al. Maternal and fetal metabonomic alterations in prenatal nicotine exposure-induced rat intrauterine growth retardation. Mol Cell Endocrinol. 2014;394:59-69.
15. Luck W, Nau H, Hansen R, Steldinger R. Extent of nicotine and cotinine transfer to the human fetus, placenta and amniotic fluid of smoking mothers. Dev Pharmacol Ther. 1985; 8:384-95. Asadi E, Jahanshahi M, Golalipour MJ. Effect of vitamin e on oocytes apoptosis in nicotine-treated mice. Iran J Basic Med Sci. 2012;15:880-4.
16. Pastrakuljic A, Schwartz R, Simone C, Derewlany LO, Knie B, Koren G. Transplacental transfer and biotransformation studies of nicotine in the human placental cotyledon perfused in vitro. Life Sci. 1998;63:2333-42.
17. Jain A, Flora SJ. Dose related effects of nicotine on oxidative injury in young, adult and old rats. J Environ Biol. 2012;33:233-8.
18. Asadi E, Jahanshahi M, Golalipour MJ. Effect of vitamin e on oocytes apoptosis in nicotine-treated mice. Iran J Basic Med Sci. 2012;15:880-4.
19. Zeidler R, Albermann K, Lang S. Nicotine and apoptosis. Apoptosis. 2007;12:1927-43.
20. Hirata N, Sekino Y, Kanda Y. Nicotine increases cancer stem cell population in MCF-7 cells. Biochem Biophys Res Commun. 2010;403:138-43.
21. Wright SC, Zhong J, Zheng H, Larrick JW. Nicotine inhibition of apoptosis suggests a role in tumor promotion. FASEB J. 1993;7:1045-51.
22. Bruin JE, Gerstein HC, Holloway AC. Long-term consequences of fetal and neonatal nicotine exposure: a critical review. Toxicol Sci. 2010;116:364-74.
23. Hutz K, Mejias-Luque R, Farsakova K, Ogris M, Krebs S, Anton M et al. The stem cell factor SOX2 regulates the tumorigenic potential in human gastric cancer cells. Carcinogenesis. 2014;35:942-50.
24. Huang J, Arsenault M, Kann M, Lopez-Mendez C, Saleh M, Wadowska D et al. The transcription factor Sry-related HMG box-4 (SOX4) is required for normal renal development in vivo. Dev Dyn. 2013;242:790-9.
25. Liu K, Lin B, Zhao M, Yang X, Chen M, Gao A et al. The multiple roles for Sox2 in stem cell maintenance and tumorigenesis. Cell Signal. 2013;25:1264-71.
26. Bareiss PM, Paczulla A, Wang H, Schairer R, Wiehr S, Kohlhofer U et al. SOX2 expression associates with stem cell state in human ovarian carcinoma. Cancer Res. 2013;73:5544-55.
27. Shen R, Pan S, Qi S, Lin X, Cheng S. Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 in gastric cancer. Biochem Biophys Res Commun. 2010;394:1047-52.
28. Prior HM, Walter MA. SOX genes: architects of development. Mol Med. 1996;2:405-12.
29. Bishayee A. Cancer prevention and treatment with resveratrol: from rodent studies to clinical trials. Cancer Prev Res (Phila). 2009;2:409-18.
30. Ahmad N, Adhami VM, Afaq F, Feyes DK, Mukhtar H. Resveratrol causes WAF-1/p21-mediated G(1)-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinom