A549 insan akciğer adenokarsinom hücre hattında rna temelli p53 tümör supresör gen aktivasyonu ve kras proto-onkogen inhibisyonunun kombine olarak terapötik kullanımının araştırılması
Küçük Resim Yok
Tarih
2023
Yazarlar
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Yayıncı
İnönü Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Amaç: Çalışmada, mutant KRAS ve yabani tip p53 taşıyan kanser hücrelerinde KRAS ekspresyonunu inhibe eden siRNA ile p53 ekpresyonunu aktive eden saRNA'nın birlikte kullanımının ayrı ayrı transfeksiyonlarına kıyasla antikanser etkisinin ortaya konması ve yeni bir RNA temelli terapötik yaklaşım geliştirilmesi amaçlanmıştır. Materyal ve Metot: Çalışmada temel olarak A549 hücre hattı ve kontrol olarak HCT116 hücre hattı kullanıldı. AuNP sentezi gerçekleştirildi, sentezlenen AuNP'lerin küçük RNA'lar ile konjugasyonları yapıldı ve hücreler konjugatlar ile transfekte edildi. Transfeksiyon sonrası mRNA ve protein ekspresyonu analizleri sırasıyla RT-qPCR ve Western Blot yöntemleri ile gerçekleştirildi. mRNA ve protein analizleri ile etkili küçük RNA konsantrasyonları belirlendi. Belirlenen konsantrasyonlar kullanılarak kombine ve ayrı ayrı olarak transfeksiyon işlemleri gerçekleştirildi ve hücre canlılığı testi, hücre döngüsü analizi, apoptoz ve nekroz testi, koloni oluşturma deneyi, yara iyileşmesi deneyi, invazyon ve migrasyon deneyleri yapılarak tranfeksiyonun neden olduğu hücresel düzeyde olaylar incelendi. Student's t-test uygulanarak sonuçlar istatistiksel olarak değerlendirildi. Bulgular: Her iki küçük RNA'nın birlikte 50 nM konsantrasyonla transfekte edilmesi, ayrı ayrı transfeksiyonlarına kıyasla sırasıyla A549 ve HCT116 hücre hatlarında p53 mRNA seviyelerinde (p<0.001, p<0.01) ve protein seviyelerinde (p<0.001, p<0.01) önemli bir artışa neden olduğu ve KRAS mRNA ve protein seviyelerini değiştirmediği saptandı (p>0.05). Her iki hücre hattında, kombine tedavi yaklaşımının ayrı ayrı transfeksiyonlara kıyasla hücre döngüsünü G0/G1 fazında daha fazla durdurduğu, apoptozu daha çok tetiklediği ve hücre proliferasyonunu, koloni oluşturma yeteneğini, invazyonu ve migrasyonu daha fazla azalttığı belirlendi. Sonuç: Çalışmamıza konu olan kombine yaklaşımın her iki hücre hattı için kontrol grubuna kıyasla antikanser etki sağladığı ve bu yaklaşımın her iki küçük RNA'nın ayrı ayrı sağlamış olduğu antikanser etkiye oranla çok daha fazla antikanser etkiye sahip olduğu ortaya konmuştur. Anahtar Kelimeler: A549, HCT116, siRNA, saRNA, KRAS, p53.
Aim: The study aimed to reveal the anticancer effect of the combined use of siRNA, which inhibits KRAS expression, and saRNA, which activates p53 expression, in cancer cells carrying mutant KRAS and wild-type p53, compared to their separate transfections, and to develop a new RNA-based therapeutic approach. Materials and Methods: In the study, A549 cell line was used as the basis and HCT116 cell line was used as a control. AuNP synthesis was performed, the synthesized AuNPs were conjugated with small RNAs and the cells were transfected with the conjugates. Post-transfection mRNA and protein expression analyzes were performed by RT-qPCR and Western Blot methods, respectively. Effective small RNA concentrations were determined by mRNA and protein analyses. Combined and separate transfection processes were carried out using the determined concentrations, and the events at the cellular level caused by transfection were examined by performing cell viability test, cell cycle analysis, apoptosis and necrosis test, colony formation experiment, wound healing experiment, invasion and migration experiments. The results were evaluated statistically by applying Student's t-test. Results: Co-transfection of small RNAs at a concentration of 50 nM was found to result in a significant increase in p53 mRNA levels (p<0.001, p<0.01) and protein levels (p<0.001, p<0.01), and did not alter KRAS mRNA and protein levels (p>0.05) in the A549 and HCT116 cell lines, respectively, compared to their individual transfections. In both cell lines, it was determined that the combined treatment approach more effectively arrested the cell cycle at the G0/G1 phase, triggered apoptosis to a greater extent, and significantly reduced cell proliferation, colony formation ability, invasion, and migration compared to individual transfections. Conclusion: It has been demonstrated that the combined approach subject to our study provides an anticancer effect for both cell lines compared to the control group, and that this approach has a much greater anticancer effect than the anticancer effect provided by both small RNAs separately. Key Words: A549, HCT116, siRNA, saRNA, KRAS, p53.
Aim: The study aimed to reveal the anticancer effect of the combined use of siRNA, which inhibits KRAS expression, and saRNA, which activates p53 expression, in cancer cells carrying mutant KRAS and wild-type p53, compared to their separate transfections, and to develop a new RNA-based therapeutic approach. Materials and Methods: In the study, A549 cell line was used as the basis and HCT116 cell line was used as a control. AuNP synthesis was performed, the synthesized AuNPs were conjugated with small RNAs and the cells were transfected with the conjugates. Post-transfection mRNA and protein expression analyzes were performed by RT-qPCR and Western Blot methods, respectively. Effective small RNA concentrations were determined by mRNA and protein analyses. Combined and separate transfection processes were carried out using the determined concentrations, and the events at the cellular level caused by transfection were examined by performing cell viability test, cell cycle analysis, apoptosis and necrosis test, colony formation experiment, wound healing experiment, invasion and migration experiments. The results were evaluated statistically by applying Student's t-test. Results: Co-transfection of small RNAs at a concentration of 50 nM was found to result in a significant increase in p53 mRNA levels (p<0.001, p<0.01) and protein levels (p<0.001, p<0.01), and did not alter KRAS mRNA and protein levels (p>0.05) in the A549 and HCT116 cell lines, respectively, compared to their individual transfections. In both cell lines, it was determined that the combined treatment approach more effectively arrested the cell cycle at the G0/G1 phase, triggered apoptosis to a greater extent, and significantly reduced cell proliferation, colony formation ability, invasion, and migration compared to individual transfections. Conclusion: It has been demonstrated that the combined approach subject to our study provides an anticancer effect for both cell lines compared to the control group, and that this approach has a much greater anticancer effect than the anticancer effect provided by both small RNAs separately. Key Words: A549, HCT116, siRNA, saRNA, KRAS, p53.
Açıklama
Anahtar Kelimeler
Tıbbi Biyoloji, Medical Biology
