In vitro yaşa bağlı makula dejenerasyonu modelinde bevacizumab ve miRNa-184'ün anjiyogenez yolağında etkinliğinin karşılaştırılması ve AnjiyomiRNA-155'in biyomarker özelliğinin araştırılması
Küçük Resim Yok
Tarih
2020
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışma ileri evre neovasküler YBMD'de yeni bir terapötik strateji olacağını düşündüğümüz miRNA-184 ile klinikte kullanım endikasyonu olan ve anti-VEGF etkisi iyi bilinen Bevacizumabın antianjiyojenik etkilerini kıyaslamak amacıyla tasarlanmıştır. Ayrıca çalışmamızda miR-184'ün anjiyogenez yolağı üzerindeki etkileri ve anjyogenezi indüklediği düşünülen miR-155'in biyomarker olabilme özelliği araştırılmıştır. Bu amaçla hipoksi ve oksidatif stres oluşturularak ARPE-19 hücre hatlarında in vitro bir YBMD modeli oluşturuldu. Hücrelerin canlılık analizi MTT testi ile yapıldı. Kullanılan miR-184 ve Bevacizumab'ın apoptotik etkileri TALİ görüntü tabanlı sitometri analizi ile değerlendirildi. Apoptoz indüksiyonu sonrası oluşan nükleer morfoloji değişiklikleri ve apoptotik yapılar moleküler flöresan görüntüleme teknikleri ile değerlendrildi. miR-184 ve Bevacizumab'ın anjiyogenez yolağı, antioksidan sistem, DNA tamir genleri, ısı şok proteinleri, hücre siklus düzenleyicileri, intrinsik ve ekstrinsik apoptoz yolağı genlerinin ekspresyonları üzerindeki etkileri qRT-PCR ile değerlendirildi. İstatistiksel analizde tek yönlü ANOVA testi, Tukey testi ve Pearson Korelasyon analizi kullanıldı. Çalışmamızda miR-184'ün HIF-1? ve HIF-1?'yı baskılayarak antianjiyojenik etki gösterdiği, Bevacizumab'ın ise bu gen üzerinden etki göstermediği ortaya konmuştur. Ayrıca miR-184'ün VEGF gen ekspresyonunu YBMD ve YBMD-BV gruplarına göre anlamlı ölçüde azalttığı gösterilmiştir. Anjiyogenezde etkili olduğu düşünülen PI3K/AKT/mTOR yolağı üzerinde miR-184'ün Bevacizumaba göre anlamlı olarak daha etkili olduğu gösterilmiştir. Pearson korelasyon analizi ile miR-184 ile HIF-1? (r= -0,448, p=0,025) ve AKT (r= -0,415, p=0,039) genleri arasında negatif korelasyon olduğu ortaya konularak bu bulgular desteklenmiştir. Bevacizumab ve miR-184'ün antioksidan sistem, DNA tamir genleri, ısı şok preoteinleri, intrinsik ve ekstrinsik apoptoz yolağında görevli genlerin ekspresyonları üzerindeki etkileri benzer saptanmış olup, ilaçların güvenlik profillerinin de benzer olduğu gösterilmiştir. MiR-155 ile anjiyogenez yolunda incelenen tüm genler istatistiksel olarak anlamlı pozitif korelasyon gösterdi. MiR-155 gen ekspresyon seviyesi incelendiğinde, YBMD-miR-184 grubunun miR-155'in en az eksprese edildiği grup olduğu bulundu ve miR-184 ve miR-155 arasındaki ters etkileşim gösterildi. Sonuç olarak çalışmamızda miR-184'ün antianjiyojenik etkisi olduğu ve bu etkisini hangi yolaklar aracılığıyla gerçekleştirdiği gösterilmiştir. Bevacizumab gibi klinik karşılığı olan bir ilaçla kıyaslayamamıza rağmen miR-184'ten alınan anlamlı sonuçlar miR-184'ün yeni bir terapötik hedef olarak sunulması olasılığının yüksek olduğunu ortaya koymuştur. Çeşitli yolaklara ait mekanizmaların çalışılması YBMD'ye dair patogenezi anlamamıza da katkıda bulunmuştur. miR-184 ile miR-155 arasındaki ters etkileşim tanı koyma ve tedaviye alınan cevap açısından miR-155'in biyomarker olabilme potansiyelini de ortaya koymaktadır.
This study was designed to compare the anti-angiogenic effects of miRNA-184 which we think will be a new therapeutic strategy in advanced neovascular AMD and Bevacizumab whose anti-VEGF effect is well known and indicated for clinical use. In addition, the effects of miR-184 on angiogenesis pathway and the biomarker feature of miR-155, which is thought to induce angiogenesis, were investigated. For this purpose, hypoxia and oxidative stress were constituted in ARPE-19 cell lines to establish an in vitro model of AMD. The viability analysis of the cells was done by MTT test. The apoptotic effects of miR-184 and Bevacizumab were evaluated by TALI image based cytometry analysis. Nuclear morphology changes and apoptotic structures formed after apoptosis induction were evaluated by molecular fluorescent imaging techniques. The effects of miR-184 and Bevacizumab on angiogenesis pathway, antioxidant system, DNA repair genes, heat shock proteins, cell cycle regulators, expression of the intrinsic and extrinsic apoptosis pathway genes were evaluated by qRT-PCR. One way ANOVA test, Tukey test and Pearson Correlation analysis were used in statistical analysis. In our study, we found that miR-184 shows antiangiogenic effects by suppressing HIF-1? and HIF-1? and Bevacizumab does not act on this gene. In addition, miR-184 has been shown to significantly reduce VEGF gene expression compared to the AMD and AMD-BV groups. MiR-184 has been shown to be significantly more effective than Bevacizumab on the PI3K / AKT / mTOR pathway, which is thought to be effective in angiogenesis. These findings were supported by suggesting a negative correlation between miR-184 and HIF-1? (r = -0,448, p = 0,025) and AKT (r = -0,415, p = 0,039) genes by Pearson correlation analysis. The effects of Bevacizumab and miR-184 on the antioxidant system, DNA repair genes, heat shock preoteins, expressions of genes involved in the intrinsic and extrinsic apoptosis pathways have been found similar, and also the safety profiles of the drugs have been shown to be similar. All the genes studied in the path of angiogenesis with miR-155 showed a statistically significant positive correlation. When the miR-155 gene expression level was examined, the AMD-miR-184 group was found the miR-155 least expressed group and the inverse interaction between miR-184 and 155 was shown. As a result, it was shown in our study that miR-184 has antiangiogenic effect and through which pathways it performs this effect. Although we could not compare with a clinically equivalent drug like Bevacizumab, significant results from miR-184 revealed that miR-184 is likely to be presented as a new therapeutic target. Studying the mechanisms of various pathways also contributed to our understanding of the pathogenesis of AMD. The inverse interaction between miR-184 and miR-155 also reveals the potential of miR-155 to become a biomarker in terms of diagnosis and response to treatment.
This study was designed to compare the anti-angiogenic effects of miRNA-184 which we think will be a new therapeutic strategy in advanced neovascular AMD and Bevacizumab whose anti-VEGF effect is well known and indicated for clinical use. In addition, the effects of miR-184 on angiogenesis pathway and the biomarker feature of miR-155, which is thought to induce angiogenesis, were investigated. For this purpose, hypoxia and oxidative stress were constituted in ARPE-19 cell lines to establish an in vitro model of AMD. The viability analysis of the cells was done by MTT test. The apoptotic effects of miR-184 and Bevacizumab were evaluated by TALI image based cytometry analysis. Nuclear morphology changes and apoptotic structures formed after apoptosis induction were evaluated by molecular fluorescent imaging techniques. The effects of miR-184 and Bevacizumab on angiogenesis pathway, antioxidant system, DNA repair genes, heat shock proteins, cell cycle regulators, expression of the intrinsic and extrinsic apoptosis pathway genes were evaluated by qRT-PCR. One way ANOVA test, Tukey test and Pearson Correlation analysis were used in statistical analysis. In our study, we found that miR-184 shows antiangiogenic effects by suppressing HIF-1? and HIF-1? and Bevacizumab does not act on this gene. In addition, miR-184 has been shown to significantly reduce VEGF gene expression compared to the AMD and AMD-BV groups. MiR-184 has been shown to be significantly more effective than Bevacizumab on the PI3K / AKT / mTOR pathway, which is thought to be effective in angiogenesis. These findings were supported by suggesting a negative correlation between miR-184 and HIF-1? (r = -0,448, p = 0,025) and AKT (r = -0,415, p = 0,039) genes by Pearson correlation analysis. The effects of Bevacizumab and miR-184 on the antioxidant system, DNA repair genes, heat shock preoteins, expressions of genes involved in the intrinsic and extrinsic apoptosis pathways have been found similar, and also the safety profiles of the drugs have been shown to be similar. All the genes studied in the path of angiogenesis with miR-155 showed a statistically significant positive correlation. When the miR-155 gene expression level was examined, the AMD-miR-184 group was found the miR-155 least expressed group and the inverse interaction between miR-184 and 155 was shown. As a result, it was shown in our study that miR-184 has antiangiogenic effect and through which pathways it performs this effect. Although we could not compare with a clinically equivalent drug like Bevacizumab, significant results from miR-184 revealed that miR-184 is likely to be presented as a new therapeutic target. Studying the mechanisms of various pathways also contributed to our understanding of the pathogenesis of AMD. The inverse interaction between miR-184 and miR-155 also reveals the potential of miR-155 to become a biomarker in terms of diagnosis and response to treatment.
Açıklama
Tıpta Uzmanlık
Anahtar Kelimeler
Göz Hastalıkları, Eye Diseases ; Tıbbi Biyoloji
