İnsan akciğer epitel hücresi ve akciğer kanseri hücrelerinde borik asidin endoplazmik retikulum stresi üzerindeki rolü
Küçük Resim Yok
Tarih
2023
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
İnönü Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Amaç: Araştırma Borik asit akciğer epitel hücreleri ve akciğer kanser hücreleri modellerinde ER stresi yolağını aktif hale getiriyor mu? Western Blot ve RT-qPCR tekniğinden yararlanılarak borik asitin ER stresi üzerindeki rolünün araştırılması, protein ve mRNA ifadelenmesinin belirlenmesi amaçlanmaktadır. Materyal Metod: Bu araştırmada A549 ve BEAS2B hücreleri model olarak kullanılmıştır. Bu doğrultuda, çalışma için uygun borik asit miktarı MTT analizleriyle tespit edilmiştir. Bu çalışmanın ardı sıra uygun koşullarda kültüre edilmiş hücrelere borik asit uygulaması yapılarak RNA ve protein izolasyonları yapılarak RT-PZR ve Western Blot analiz testleri yapılmıştır. Bulgular: ER stres yolağında rol alan genlerin (PERK, XBP1, MAOA, ATF6) gen ifadelenmesi seviyeleri relatif kuantitatif analizler ve Student T testi yapılarak karşılaştırılmıştır. PERK, XBP1 genlerinde ER stresine bağlı UPR' ye cevap olarak borik asitin apoptozu tetiklediği (PERK p1=0.000488 p2=0.000977, XBP1 p1=0.000253 p2=0.000506) p<0.05 değerleri ile istatistiksel olarak anlamlı fark saptanmışken, MAOA ve ATF6 strese bağlı aşırı gen ifadelenmesi tesbit edilmemiş olup (MAOA p1=0.450321203 p2=0.900642405, ATF6 p1=0.496131 p2=0.992261) değerleri ile anlamlı fark saptanmadığı görülmüştür. İmmünblotlama analizlerinde de PERK, XBP1'de protein seviye analizlerinin kontrole oranladığında RT-PZR analiz sonuçlarını desteklediği görülmüştür. ATF6 ve MAOA için ise PZR sonuçlarına benzer anlamlı fark görülmemiştir. MAOA, RT-PZR analiz sonucuna nazaran immünblotlama analizinde anlamlı fark görülmüştür. Sonuç: Analizler sonucunda, A549 ve BEAS2B hücrelerine borik asit muamelesi kontrole kıyasla ER stresini artırdığını buna bağlı olarak PERK, XBP1 ve MAOA anlamlı bir fark görülmüş olup ATF6 'da anlamlı fark görülmemiştir. Anahtar Kelimeler: ATF6, Bor toksisitesi, Endoplazmik Retikulum(ER) stresi, Real Time qPCR, Western blot
Aim: Research Does boric acid activate the ER stress pathway in lung epithelial cells and lung cancer cell models? It is aimed to investigate the role of boric acid on ER stress and to determine protein and mRN expression by using Western Blot and RT-qPCR technique. Material and Method: A549 and BEAS2B cells were used as models in this study. In this direction, the amount of boric acid suitable for the study was determined by MTT analysis. Following this study, RNA and protein isolations were made by applying boric acid to cells cultured under appropriate conditions, and RT-PCR and Western Blot analysis tests were performed. Results: Gene expression levels of genes involved in the ER stress pathway (PERK, XBP1, MAOA, ATF6) were compared by performing relative quantitative analyzes and Student's T test. In PERK, XBP1 genes, there was a statistically significant difference with p<0.05 values in which boric acid triggered apoptosis in response to ER stress-induced UPR (PERK p1=0.000488 p2=0.000977, XBP1 p1=0.000253 p2=0.000506), while MAOA and ATF6 stress-induced excessive gene expression was not detected (MAOA p1=0.450321203 p2=0.900642405, ATF6 p1=0.496131 p2=0.992261). In immunoblotting analyzes, it was seen that protein level analyzes in PERK and XBP1 supported the results of RT-PCR analysis when compared to the control. There was no significant difference similar to PCR results for ATF6 and MAOA. A significant difference was observed in the immunoblotting analysis compared to the MAOA, RT-PCR analysis result. Conclusion: As a result of the analyzes, boric acid treatment of A549 and BEAS2B cells increased ER stress compared to the control, accordingly, a significant difference was observed in PERK, XBP1 and MAOA, but no significant difference was observed in ATF6. Key words: ATF6, Boron toxicity, Endoplasmic Reticulum(ER) stress, Real Time qPCR, Western blot
Aim: Research Does boric acid activate the ER stress pathway in lung epithelial cells and lung cancer cell models? It is aimed to investigate the role of boric acid on ER stress and to determine protein and mRN expression by using Western Blot and RT-qPCR technique. Material and Method: A549 and BEAS2B cells were used as models in this study. In this direction, the amount of boric acid suitable for the study was determined by MTT analysis. Following this study, RNA and protein isolations were made by applying boric acid to cells cultured under appropriate conditions, and RT-PCR and Western Blot analysis tests were performed. Results: Gene expression levels of genes involved in the ER stress pathway (PERK, XBP1, MAOA, ATF6) were compared by performing relative quantitative analyzes and Student's T test. In PERK, XBP1 genes, there was a statistically significant difference with p<0.05 values in which boric acid triggered apoptosis in response to ER stress-induced UPR (PERK p1=0.000488 p2=0.000977, XBP1 p1=0.000253 p2=0.000506), while MAOA and ATF6 stress-induced excessive gene expression was not detected (MAOA p1=0.450321203 p2=0.900642405, ATF6 p1=0.496131 p2=0.992261). In immunoblotting analyzes, it was seen that protein level analyzes in PERK and XBP1 supported the results of RT-PCR analysis when compared to the control. There was no significant difference similar to PCR results for ATF6 and MAOA. A significant difference was observed in the immunoblotting analysis compared to the MAOA, RT-PCR analysis result. Conclusion: As a result of the analyzes, boric acid treatment of A549 and BEAS2B cells increased ER stress compared to the control, accordingly, a significant difference was observed in PERK, XBP1 and MAOA, but no significant difference was observed in ATF6. Key words: ATF6, Boron toxicity, Endoplasmic Reticulum(ER) stress, Real Time qPCR, Western blot
Açıklama
Anahtar Kelimeler
Genetik, Genetics, Moleküler Tıp