Deneysel meme tümörü modeline bağlı gelişen karaciğer metastazında gliko-gen ifadelerinin ve glikoprotein yapılarının belirlenmesi ve biyobelirteç potansiyellerinin araştırılması
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Tarih
2023
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Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Meme kanseri, ABD'deki tüm yeni kanser vakalarının %29'unu oluşturan ve kadınlar arasında yaygın görülen kanserdir. İnvazif meme kanserinin vücuttaki diğer organlara metastaz yapması muhtemeldir. Glikanlar, proliferasyon, invazyon ve metastaz dahil olmak üzere tümör progresyonunun farklı yönlerini düzenleyebilirler. Meme kanseri ve birçok kanser türünde glikozilasyon modellerinde değişiklikler gözlenmiştir. Glikoproteinler ayrıca hastalık tespiti için biyobelirteçleri keşfetmek için ideal bir kaynak sağlamıştır. İnsan proteinlerinin %50'den fazlasının, proteinlerin işlevselliğini modüle etmek için glikozile olduğuna inanılmaktadır. Onaylanmış kanser biyobelirteçlerinin çoğu, glikoprotein yapısındadır ve bu proteinlerin sayıları çok fazla değildir. Glikoproteinlerin etkili kalitatif ve kantitatif değerlendirmesi, zenginleştirme yöntemlerini gerektirmektedir. Bu bilgiler ışığında, bu tezde Ehrlic asit ile oluşturulmuş karaciğer metastazı meme tümörü modelinde glikozilasyon ilişkili enzimlerin ekspresyon düzeyleri qRT-PCR array yöntemi ile araştırılmış ve istatistik olarak değerlendirmeleri yapılmıştır. Ayrıca kontrol ve tümör gruplarında, LC-QTOF/MS cihazı ile proteomiks çalışmaları yapılarak, biyobelirteç potansiyelleri araştırılmıştır. Işık mikroskobik incelemede; Hematoksilen-Eozin ile boyanmış kontrol ve deney grubuna ait karaciğer dokularında rutin ışık mikroskobu preparatlarında yapılan incelemelerde deney grubunda histopatolojik bulgulara rastlanmıştır. Fibrotik doku oluşumları, karaciğer hücre kordonlarında bozulma, sinüzoidal alanlarda genişleme, endotel hasarı, hepatosit ve nükleus membranlarında dejenerasyon gözlenmiştir. Hücre sınırlarının belirgin olmadığı ve hücre çekirdekleri arasında şekil/büyüklük açısından farklılıklar olduğu gözlenmiş olup hipertrofik hepatositlere rastlanmıştır. Ayrıca piknotik nükleuslar hemoralji ve nekrotik doku alanları tespit edilmiştir. Deneysel oluşturulmuş karaciğer metaztazı meme tümörü ve sağlıklı hayvanlara ait karaciğer dokularında; 192 adet gliko-genin ekspresyon analizi qRT-PCR ile gerçekleştirilmiştir. Analiz sonucunda elde etmiş olduğumuz relatif kat değişimlerini T testi yaparak istatistiksel olarak değerlendirilmiştir. T testi sonucunda kontrol ve deney grubunda; 32 adet gliko-genin anlamlı farklılık gösterdiği belirlenmiştir. Glikoproteomiks sonuçlarında ise; LCQTOF/MS cihazından elde edilen veriler, SearchGUi yazılımına yüklenmiş ve uygun kütüphaneler seçilerek proteinlerin tanımlanması yapılmıştır. Elde edilen sonuçlar PeptideShaker yazılımı ile gösterilmiştir. Toplam 14 adet glikoprotein deney ve kontrol grubunda farklı bulunmuştur. Kontrol grubunda: Alfa-2-HS-glikoprotein, Fosfogliserat mutaz 1 ve Alfa endolaz. Deney grubunda: Beta 1,3 glikoziltransferaz, Nasetilgalaktozaminiltransferaz-7, Glikoprotein endo-alfa-1,2-mannosidaz, Glikoziltransferaz 1 alan içeren protein 1, Polipeptit N-asetilgalaktozaminiltransferaz 2, Sfingomyelin fosfodiesteraz, Bifonksiyonel UDP-N-Asetilglukozamin 2-epimeraz/Nasetilmannosamin kinaz, Regukalsin, ADP/ATP translokaz 1, Sıralama nexin-9 ve Protein RFT1 homologu proteinleri saptanmıştır. Gen ekspresyonu ve proteomiks çalışmaları sonucunda 5 adet biyobelirteç adayı glikoprotein tespit edilmiştir. Bunlar; B3GLCT-1, MANEA, GNE, GALNTL2, RFT-1 proteinleridir. Bu proteinler; glikolizasyon, metastaz proliferasyon gibi biyolojik süreçlerde yer almaktadırlar. Sonuç olarak; elde ettiğimiz sonuçlar, metastazı etkileyen ve dolayısıyla potansiyel terapötik hedefleri temsil edebilen glikozilasyon değişimleri hakkında yeni bilgiler sunmaktadır. Deneysel meme tümörüne bağlı gelişen metastatik karaciğer dokusunda belirlenen glikan profilleri, glikanların biyolojik rollerinin aydınlatılmasına katkı sağlayacak olup bu yapıların biyobelirteç potansiyeli olan proteinlerin, teropatik çalışmaların yapılacağı araştırmalara önemli veri sunacağı düşülmektedir.
Women are more likely than men to develop breast cancer, which accounts for 29% of all new cancer cases in the United States. It is conceivable that invasive breast cancer may spread to other organs of the body. Among other aspects of tumor growth, glycans can control the proliferation, invasion, and metastasis of tumor cells. Many cancers, including breast cancer, have been linked to changes in glycosylation patterns. Moreover, glycoproteins have been a great source for identifying disease biomarkers. It is estimated that more than 50% of human proteins are glycosylated to alter their function. The majority of recognized cancer biomarkers are glycoproteins, which are low in protein. Enrichment techniques are required for accurate qualitative and quantitative assessment of glycoproteins. In the light of this, in this thesis, the expression levels of glycosylation-related enzymes in the liver metastasis breast tumor model induced with Ehrlic acid have been investigated by qRT-PCR array method and statistically evaluated. In addition, the potential of biomarkers was investigated by performing proteomics studies using LC-QTOF / MS device in control and tumor groups. In light microscopic examination; In the examinations made on routine light microscopy preparations in the liver tissues of the control and experimental groups stained with Hematoxylin-Eosin, histopathological findings were found in the experimental group. Fibrotic tissue formations, deterioration of liver cell cords, expansion of sinusoidal areas, endothelial damage, and degeneration of hepatocyte and nuclear membranes were observed. It was observed that cell boundaries were not clear and there were differences in shape/size between cell nuclei, and hypertrophic hepatocytes were observed. Additionally, pyknotic nuclei, hemorrhage and necrotic tissue areas were detected. Experimentally induced liver metastasis in mammary tumors and liver tissues of healthy animals; Expression analysis of 192 glyco-genes was performed by qRT-PCR. The relative fold changes we obtained as a result of the analysis were evaluated statistically by performing a T test. As a result of the t test, in the control and experimental groups; It was determined that 32 glyco-genes showed significant differences. In the glycoproteomics results; The data obtained from the LC-QTOF/MS device was uploaded to SearchGUi software and proteins were identified by selecting appropriate libraries. The results obtained are shown with PeptideShaker software. A total of 14 glycoproteins were found to be different in the experimental and control groups. In the control group: Alpha2-HS-glycoprotein, Phosphoglycerate mutase 1 and Alpha endolase. In the experimental group: Beta 1,3 glycosyltransferase, N-acetylgalactosaminyltransferase-7, Glycoprotein endo-alpha-1,2-mannosidase, Glycosyltransferase 1 domain-containing protein 1, Polypeptide N-acetylgalactosaminyltransferase 2, Sphingomyelin phosphodiesterase, Bifunctional UDP-N-Acetylglucosamine 2- epimerase/N-acetylmannosamine kinase, Regukalsin, ADP/ATP translocase 1, Sorting nexin-9 and Protein RFT1 homolog proteins were detected. As a result of gene expression and proteomics studies, 5 biomarker candidate glycoproteins were identified. These; B3GLCT-1, Manea, GNE, GALNTL2, RFT-1 proteins. In conclusion; Our results provide new insights into glycosylation alterations that affect metastasis and may thus represent potential therapeutic targets. Glycan profiles determined in metastatic liver tissue resulting from experimental breast tumor will contribute to the elucidation of the biological roles of glycans, and it is thought that the proteins with biomarker potential of these structures will provide important data for research on therapeutic studies.
Women are more likely than men to develop breast cancer, which accounts for 29% of all new cancer cases in the United States. It is conceivable that invasive breast cancer may spread to other organs of the body. Among other aspects of tumor growth, glycans can control the proliferation, invasion, and metastasis of tumor cells. Many cancers, including breast cancer, have been linked to changes in glycosylation patterns. Moreover, glycoproteins have been a great source for identifying disease biomarkers. It is estimated that more than 50% of human proteins are glycosylated to alter their function. The majority of recognized cancer biomarkers are glycoproteins, which are low in protein. Enrichment techniques are required for accurate qualitative and quantitative assessment of glycoproteins. In the light of this, in this thesis, the expression levels of glycosylation-related enzymes in the liver metastasis breast tumor model induced with Ehrlic acid have been investigated by qRT-PCR array method and statistically evaluated. In addition, the potential of biomarkers was investigated by performing proteomics studies using LC-QTOF / MS device in control and tumor groups. In light microscopic examination; In the examinations made on routine light microscopy preparations in the liver tissues of the control and experimental groups stained with Hematoxylin-Eosin, histopathological findings were found in the experimental group. Fibrotic tissue formations, deterioration of liver cell cords, expansion of sinusoidal areas, endothelial damage, and degeneration of hepatocyte and nuclear membranes were observed. It was observed that cell boundaries were not clear and there were differences in shape/size between cell nuclei, and hypertrophic hepatocytes were observed. Additionally, pyknotic nuclei, hemorrhage and necrotic tissue areas were detected. Experimentally induced liver metastasis in mammary tumors and liver tissues of healthy animals; Expression analysis of 192 glyco-genes was performed by qRT-PCR. The relative fold changes we obtained as a result of the analysis were evaluated statistically by performing a T test. As a result of the t test, in the control and experimental groups; It was determined that 32 glyco-genes showed significant differences. In the glycoproteomics results; The data obtained from the LC-QTOF/MS device was uploaded to SearchGUi software and proteins were identified by selecting appropriate libraries. The results obtained are shown with PeptideShaker software. A total of 14 glycoproteins were found to be different in the experimental and control groups. In the control group: Alpha2-HS-glycoprotein, Phosphoglycerate mutase 1 and Alpha endolase. In the experimental group: Beta 1,3 glycosyltransferase, N-acetylgalactosaminyltransferase-7, Glycoprotein endo-alpha-1,2-mannosidase, Glycosyltransferase 1 domain-containing protein 1, Polypeptide N-acetylgalactosaminyltransferase 2, Sphingomyelin phosphodiesterase, Bifunctional UDP-N-Acetylglucosamine 2- epimerase/N-acetylmannosamine kinase, Regukalsin, ADP/ATP translocase 1, Sorting nexin-9 and Protein RFT1 homolog proteins were detected. As a result of gene expression and proteomics studies, 5 biomarker candidate glycoproteins were identified. These; B3GLCT-1, Manea, GNE, GALNTL2, RFT-1 proteins. In conclusion; Our results provide new insights into glycosylation alterations that affect metastasis and may thus represent potential therapeutic targets. Glycan profiles determined in metastatic liver tissue resulting from experimental breast tumor will contribute to the elucidation of the biological roles of glycans, and it is thought that the proteins with biomarker potential of these structures will provide important data for research on therapeutic studies.
Açıklama
Anahtar Kelimeler
Meme kanseri, Metastaz, Glikoprotein, Glikogen, Glikoproteomiks, Breast cancer, Metastasis, Glycoprotein, Glycogen, Glycoproteomics
