siRNA Mediated Gene Silencing in the Pancreatic Cancer Capan-1 Cell Line

Abstract

Today, pancreatic cancer ranks second after the cardiovascular system among the causes of death. The pancreatic cancer which is an aggressive type progressing without giving too many symptoms. In addition, factors such as difficulty in early diagnosis, rapid metastasis and non-response to traditional treatments cause a low survival rate in this type of cancer. Therefore, many studies are carried out to develop alternative diagnosis and treatment methods. Focus is put on target-oriented studies to increase the survival rate and eliminate other negative effects. The gene therapy method is at the forefront of such studies. RNA interference (RNAi) molecules are crucial in this method. From among these molecules, small non-coding RNA (siRNA) are used in studies as a therapeutic agent by its delivery to the target gene by various mechanisms. Studies are carried out with different biological and chemical agents to deliver the molecules to the target cell efficiently. This study aimed to silence the c-Myc gene using gold nanoparticle (AuNP)-siRNA in the Capan-1 cell line. We performed Real-Time PCR, Dual Staining and gel electrophoresis using gold nanoparticles (2X, 4X, 8X) and siRNA (25 nM) at concentrations determined in the Capan-1 cell line. The data obtained from the analysis suggested that the nanoparticle siRNA complex could be used effectively in silencing the target gene. Nevertheless, effective results could be obtained with more knowledge on this subject.

Keywords

• gene therapy
• c-Myc
• siRNA
• Capan-1
• Real-Time PCR
• pancreatic cancer

References

1. [1]. http://gco.iarc.fr/today (Date Accessed: 08.12.2021).
2. [2]. Siegel RL, Miller KD, and Jemal A.. Cancer statistics, 2015. CA Cancer J. Clin. 2015; 65: 5–29. doi: 10.3322/caac.21254.
3. [3]. Schottenfeld D. and Fraumeni JR. Cancer epidemiology and prevention: Oxford University Press; 2006.
4. [4]. Zhou L, Sawaguchi S, Twining SS, Sugar J, Feder RS, Yue BY.. Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci. 1998; 39 (7): 1117-24.
5. [5]. Kenney MC, Chwa M, Lin B, Huang GH, Ljubimov AV, Brown DJ. Identification of cell types in human diseased corneas. Cornea. 2001; 20 (3): 309-16.
6. [6]. Parkin BT, Smith VA, Easty DL. The control of matrix metalloproteinase-2 expression normal and keratoconic corneal keratocyte cultures. Eur J Ophthalmol. 2000; 10(4): 276-85.
7. [7]. Buchholz M, SchatzMartin A, Wagner M, Michl P, Linhart T, Adler G, Gress TM, Ellenrieder V. Overexpression of c-myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway, EMBO J. 2006; 25: 3714–3724.
8. [8]. Buchholz M, SchatzMartin A, Wagner M, Michl P, Linhart T, Adler G, Gress TM, Ellenrieder V. Overexpression of c-myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway, EMBO J. 2006; 25: 3714–3724.

9. [9]. Miller DM, Thomas SD, Islam A, Muench D, Sedoris K. c-Myc and cancer metabolism. Clin. Cancer Res. 2012; 18: 5546–5553.
10. [10]. Meng XW, Lee SH, Kaufmann SH. Apoptosis In The Treatment Of Cancer: A Promise Kept?. Curr Opin Cell Biol, 2006; 18: 668-76.
11. [11]. Kim WJ, Shah S, Wilson SE. Differences in keratocyte apoptosis following transepithelial and laser - scrape photorefractive keratectomy in rabbits. J Refract Surg. 1998; 14(5):526-33.
12. [12]. Wilson SE. Role of apoptosis in wound healing in the cornea. Cornea. 2000; 19:7-12.
13. [13]. Grusch M, Fritzer-Szekeres M, Fuhrmann G, Rosenberger G, Luxbacher C, Elford HL, Smid K, Peters GJ, Szekeres T, Krupitza G. Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox. Experimental Hematology. 2001; 29(5): 623-632.
14. [14]. Fahrig R, Heinrich JC, Nickel B, Wilfert F, Leisser C, Krupitza G, Praha C, Sonntag D, Fiedler B, Scherthan H, Ernst H. Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2'- deoxyuridine (RP101). Cancer Research.2003; 63(18): 5745-5753.
15. [15]. Gad SC. Handbook of pharmaceutical biotechnology. Vol. 2: John Wiley & Sons. 2007.
16. [16]. Giacca M, Zacchigna S. Virus-mediated gene delivery for human gene therapy. Journal of Controlled Release. 2012; 161: 377–388.
17. [17]. Gad, SC, Handbook of pharmaceutical biotechnology. John Wiley & Sons. 2007, Vol. 2.
18. [18]. Daniel S. Advanced textbook on gene transfer, gene therapy and genetic pharmacology: principles, delivery and pharmacological and biomedical applications of nucleotide-based therapies. 2013, World Scientific: Vol. 1.
19. [19]. Hosseinkhani H, Domb AJ. Biodegradable polymers in gene‐silencing technology. Polym Adv Technol. 2019; 30:2647–2655. [20]. Hosseinkhani H, Chen YR, He W, Hong PD, Yu DS, Domb AJ. Engineering of magnetic DNA nanoparticles for tumor‐targeted therapy. J Nanopart Res. 2013; 15(1):1345‐1355.
20. [21]. Gündoğdu R, Çelik V. RNA İnterferans (RNAi). Erciyes Üniversitesi Fen Bilimleri Enstitüsü Fen Bilimleri Dergisi, 2009; 25(1): 34-47.
21. [22]. Aagaard L, Rossi JJ. RNAi therapeutics: Principles, prospects and challenges. Adv Drug Deliv Rev. 2007; 59: 75–86.
22. [23]. Attar A. Gen terapisi yöntemleri: fiziksel ve kimyasal metotlar. Turkish Bulletin of Hygiene & Experimental Biology/Türk Hijyen ve Deneysel Biyoloji. 2017; 74(1):103-112.
23. [24]. Sayıner Ö, Çomoğlu T. Nanotaşıyıcı sistemlerde hedeflendirme. Ankara Üniversitesi Eczacılık Fakültesi Dergisi. 2016; 40(3): 62-79.
24. [25]. Adijanto J., Naash M.I. 2015. Nanoparticle-based technologies for retinal gene therapy. Eur J Pharm Biopharm, 95: 353-67.
25. [26]. Alexis F. Nanoparticle technologies for cancer therapy, in Drug delivery. Springer. 2010; 197: 55-86.
26. [27]. Bhattacharya R, Mukherjee P. Biological properties of "naked" metal nanoparticles. Adv Drug Deliv Rev. 2008; 60(11): 1289-306.
27. [28]. Urie R, Rege K. Nanoscale inorganic scaffolds as therapeutics and delivery vehicles. Current Opinion in Chemical Engineering. 2015; 7: 120-128.
28. [29]. Dykman L. Khlebtsov NGold nanoparticles in biomedical applications: recent advances and perspectives. Chemical Society Reviews. 2012; 41(6):2256-2282.
29. [30]. Daniels AN, Singh M. Sterically stabilized siRNA:gold nanocomplexes enhance c-MYC silencing in a breast cancer cell model. Nanomedicine. 2019; 14: 1387-1401.
30. [31]. Ku SH, Kim K, Choi K, Kim SH, Kwon I.C. Tumor-Targeting Multifunctional Nanoparticles for siRNA Delivery: Recent Advances in Cancer Therapy. Adv. Healthcare Mater. 2014; 3: 1182–1193.
31. [32]. Shaat H., Mostafa A, Moustafa M, Gamal-Eldeen A, Emam A, El-Hussieny E, Elhefnawi M. Modified gold nanoparticles for intracellular delivery of anti-liver cancer siRNA. International journal of pharmaceutics. 2016; 504: 125-133.