Küçük hücreli dışı akciğer kanserinde cisplatin direnci gelişiminde rol oynayan genetik faktörlerin CRISPR-CAS9 tabanlı tüm genom tarama sistemiyle belirlenmesi
Küçük Resim Yok
Dosyalar
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Yüksek metastatik etkisi ile tanımlanan akciğer kanseri, en sık teşhis edilen kanser türüdür. Maligniteyi kontrol etmek için kullanılan başlıca tedavi yöntemi kemoterapidir. Kemoterapötikler arasında yer alan cisplatin, akciğer kanseri dahil birçok kanser türünün tedavisinde kullanılan platin bazlı bir ajandır. Küçük hücreli dışı akciğer kanserinin (KHDAK) standart tedavisinde kullanılan cisplatine karşı olguların ~%60'ının zamanla direnç geliştirdikleri bilinmektedir. Bu yüzden cisplatin direnç mekanizmasının aydınlatılması ve yeni moleküler hedeflerin belirlenmesi gerekmektedir. Bu tez calışmasında genom çapında CRISPR-Cas9 -tabanlı genetik tarama yöntemiyle cisplatine duyarlı ve dirençli KHDAK hücre serisi Calu-1'in bağımlı olduğu bileşenlerin belirlenmesi ve bu bileşenlerin kanser tedavisinde kullanılabilirliklerinin araştırılması amaçlanmıştır. Çalışmada, CRISPR-Cas9 -tabanlı genetik tarama için 19,114 insan genini hedefleyen ve toplam 76,441 guide RNA'dan (gRNA) oluşan lentiviral 'Brunello kütüphanesi' kullanıldı. Calu-1 hücreleri 'doz arttırımı' metoduyla cisplatine karşı 6.3-kat dirençli hale getirildikten sonra cisplatine duyarlı ve dirençli Calu-1 hücreleri kütüphane ile transfekte edildi. Transfeksiyondan sonra gruplar arasındaki gRNA dağılımının belirlenmesi için yeni nesil dizi analizi (NGS) ile amplikon sekanslama yaptırıldı. NGS sonuçları biyoinformatik analizlerden geçirilerek her hücre grubunda artan ve azalan gRNA'lar belirlenerek bu gRNA'ların hedefledikleri genler listelendi. Aday genler, dirençli hücrede cisplatin duyarlılığını artırmak için azalan gRNA'ların hedefledikleri genler arasından seçildi. Azalan genlerin hücre döngüsü, protein veya lipid metabolizması, vezikül, protein veya iyon transportu, DNA tamiri, mRNA splicing, gen ekspresyonu, RNA işlenmesi, epigenetik, apoptoz, protein modifikasyonu gibi önemli biyolojik yolaklarda rol oynadıkları belirlendi. Dirençli Calu-1 hücrelerinde gen skoruna ve akciğer kanserindeki yüksek ekspresyon seviyesine göre aday gen olarak GPR89A seçildi. Bu aday gen dirençli Calu-1 hücrelerinde knock-out edildikten sonra hücre canlılığı, sitotoksisite, apoptoz, hücre döngüsü, koloni oluşumu, migrasyon gibi önemli hücresel fonksiyonlar ile ATF4 gen ekspresyonu üzerine olan etkileri fonksiyonel testlerle belirlendi. Yapılan fonksiyonel analizler sonucunda GPR89A knock-out dirençli Calu-1 hücrelerinin cisplatin duyarlılığının arttığı, cisplatin tedavisi sonrası apoptozunun indüklendiği, hücre döngüsünün S evre arresti sonucu mitotik katastrofa neden olduğu, koloni oluşumunu ve migrasyonunu engellediği belirlendi. Bu çalışma ile cisplatin dirençli KHDAK hücrelerinde ilk kez genom çapında tarama yapılarak GPR89A geninin direnç gelişiminden sorumlu bir faktör olduğu ortaya konmuştur.
Lung cancer, which is defined by its high metastatic effect, is the most frequently diagnosed cancer type. The main treatment method used to control malignancy is chemotherapy. Cisplatin, which is among the chemotherapeutics, is a platinum-based agent used in the treatment of many types of cancer, including lung cancer. It is known that ~60% of cases develop resistance to cisplatin, which is used in the standard treatment of non-small cell lung cancer (NSCLC). Therefore, it is necessary to elucidate the mechanism of cisplatin resistance and to identify new molecular targets. In this thesis, it was aimed to determine the dependent components of the cisplatin-sensitive and resistant NSCLC cell line Calu-1 with a genome-wide CRISPR-Cas9-based genetic screening method and to investigate the usability of these components in cancer therapy. The study used a lentiviral "Brunello library" consisting of a total of 76,441 guide RNAs (gRNAs) targeting 19,114 human genes for CRISPR-Cas9-based genetic screening. After the Calu-1 cells were rendered 6.3-fold resistant to cisplatin by 'dose escalation' method, cisplatin-sensitive and resistant Calu-1 cells were transfected with the library. After transfection, deep sequencing was performed with NGS to determine the distribution of gRNA between cell groups. NGS results were analyzed by bioinformatics, increasing and decreasing gRNAs in each cell group were determined and the genes targeted by these gRNAs were listed. Candidate genes were selected from among the genes targeted by declining gRNAs to increase cisplatin sensitivity in resistant cells. Decreased genes were determined to play a role in important biological pathways such as cell cycle, protein or lipid metabolism, vesicle, protein or ion transport, DNA repair, mRNA splicing, gene expression, RNA processing, epigenetics, apoptosis, and protein modification. In resistant Calu-1 cells, GPR89A was selected as the candidate gene based on gene score and high expression level in lung cancer. After this candidate gene was knocked out in resistant Calu-1 cells, its effects on important cellular functions such as cell viability, cytotoxicity, apoptosis, cell cycle, colony formation, migration and ATF4 gene expression were determined by functional tests. As a result of functional analysis, it could be determined that cisplatin sensitivity of GPR89A knockout resistant Calu-1 cells was increased, apoptosis was induced after cisplatin treatment, mitotic catastrophe as a result of S-stage arrest of the cell cycle was caused, and colony formation and migration was prevented. In conclusion, with this study, genetic factors responsible for the development of cisplatin resistance in lung cancer were determined for the first time, and it was determined that the GPR89A gene was the dependent component responsible for cisplatin resistance in NSCLC cells.
Lung cancer, which is defined by its high metastatic effect, is the most frequently diagnosed cancer type. The main treatment method used to control malignancy is chemotherapy. Cisplatin, which is among the chemotherapeutics, is a platinum-based agent used in the treatment of many types of cancer, including lung cancer. It is known that ~60% of cases develop resistance to cisplatin, which is used in the standard treatment of non-small cell lung cancer (NSCLC). Therefore, it is necessary to elucidate the mechanism of cisplatin resistance and to identify new molecular targets. In this thesis, it was aimed to determine the dependent components of the cisplatin-sensitive and resistant NSCLC cell line Calu-1 with a genome-wide CRISPR-Cas9-based genetic screening method and to investigate the usability of these components in cancer therapy. The study used a lentiviral "Brunello library" consisting of a total of 76,441 guide RNAs (gRNAs) targeting 19,114 human genes for CRISPR-Cas9-based genetic screening. After the Calu-1 cells were rendered 6.3-fold resistant to cisplatin by 'dose escalation' method, cisplatin-sensitive and resistant Calu-1 cells were transfected with the library. After transfection, deep sequencing was performed with NGS to determine the distribution of gRNA between cell groups. NGS results were analyzed by bioinformatics, increasing and decreasing gRNAs in each cell group were determined and the genes targeted by these gRNAs were listed. Candidate genes were selected from among the genes targeted by declining gRNAs to increase cisplatin sensitivity in resistant cells. Decreased genes were determined to play a role in important biological pathways such as cell cycle, protein or lipid metabolism, vesicle, protein or ion transport, DNA repair, mRNA splicing, gene expression, RNA processing, epigenetics, apoptosis, and protein modification. In resistant Calu-1 cells, GPR89A was selected as the candidate gene based on gene score and high expression level in lung cancer. After this candidate gene was knocked out in resistant Calu-1 cells, its effects on important cellular functions such as cell viability, cytotoxicity, apoptosis, cell cycle, colony formation, migration and ATF4 gene expression were determined by functional tests. As a result of functional analysis, it could be determined that cisplatin sensitivity of GPR89A knockout resistant Calu-1 cells was increased, apoptosis was induced after cisplatin treatment, mitotic catastrophe as a result of S-stage arrest of the cell cycle was caused, and colony formation and migration was prevented. In conclusion, with this study, genetic factors responsible for the development of cisplatin resistance in lung cancer were determined for the first time, and it was determined that the GPR89A gene was the dependent component responsible for cisplatin resistance in NSCLC cells.
Açıklama
Anahtar Kelimeler
KHDAK, CRISPR-Cas9, Cisplatin Direnci, Genetik Tarama, GPR89A, NSCLC, CRISPR-Cas9, Cisplatin Resistance, Genetic Screening, GPR89A
