Mide kanserinde matriks metalloproteinaz (MMP) genlerinin ifadesi üzerine oksidatif stres ve RNA müdahalesinin etkisi
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Dosyalar
Tarih
2009
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
İnönü Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Mide kanseri dünya genelinde en sık görülen dördüncü kanser türü olup son yıllarda görülme sıklığının azalmasına rağmen, kanserle ilişkili ölüm nedenlerinin başında gelmektedir. Kanserin ortaya çıkmasında oksidatif stresin rol oynadığı her ne kadar bilinse de, bu ilişkinin nasıl olduğu çok iyi bilinmemektedir. Tümör hücrelerinin invazyon ve metastaz yapmaları, bölgesel bağ dokuyu parçalayan matriks metalloproteinazlar (MMP) ile gerçekleşir. Tümör hücrelerinin daha agresif hale gelmesinde ve genel anlamda kanserin yayılmasında MMP'ler büyük öneme sahiplerdir. Bu çalışmada, tümör invazyonunda rol oynayan MMP genlerinin ve onkojenik transformasyonda rol oynayan ß-katenin'in gastrik kanser biyopsilerinde ve hücre hatlarında oksidatif strese bağlı olarak değişimlerinin gösterilmesi amaçlanmıştır. AGS hücre hattında MMP-3'e özgü RNA müdahalesinin etkisini araştırmak çalışmanın diğer bir amacını oluşturmaktadır. Gastrik kanserli biyopsi örneklerinde gen ifadelerine RT PCR, hücre hatlarında stres koşulları altında değişen gen ifadelerine ise Q-RT PCR ile bakılmıştır. Doku örnekleri ve hücre hatlarında ß-katenin ve 8-OHdG'in lokalizasyonuna immünohistokimyasal yöntemle bakılmıştır. Değişen stres koşullarında hücre hatlarının invazyon seviyelerine bakılmıştır. MMP-3'ün ifade olduğu AGS hücre hattında MMP-3 siRNA'sı hücre içerisine transfeksiyon yöntemi ile sokulup gen ifadesi değişimlerine Q-RT PCR ile bakılmıştır. Çalışılan doku örneklerinde MMP ve ß-katenin geninlerinin tümörlü dokularda daha fazla ifade olduğu görülmüştür. Hücre içinde ß-katenin'in % 85 oranında sitoplazmik ve membranöz lokalizasyonu görülürken, % 20 oranında nükleer lokalizasyonu görülmüştür. Hücre hatlarında H2O2'nin göstermiş olduğu toksisite benzerlik gösterirken, hücrelerin bu strese verdikleri tepki farklı olmuştur. MKN-45 hücre hattı H2O2 ile apoptoza girerken diğer hücre hatlarının girmediği görülmüştür. H. pylori enfeksiyonu'nun ß-katenin ve MMP genlerinin ifadelerinde değişiklik yaratmazken H2O2 MMP-1, MMP-7, MMP-14, MMP-15, MMP-17 ve ß-katenin ifadesinde artışa sebep olmuştur. Stres koşulunun ortadan kaldırıldığı durumda gen ifadelerinde görülen azalış, gen ifadelerindeki değişmenin tamamen oksidatif strese bağlı olduğunun göstergesidir. AGS hücre hattında MMP-3 genin susturulması MMP-15 ifadesini azaltırken MMP-10'nun ifadesini arttırmıştır. Gastrik kanserde de MMP-3 genin diğer MMP'leri regüle etmesiyle kilit bir göreve sahip olabileceği düşünülmektedir.
Gastric cancer is the fourth leading cancer type of all cancers and although there has been a decline in the incidence of this cancer in recent years, it is still one of the most common cause of deaths. Although the role of oxidative stress on cancer formation has long been known, its mechanism has not been elucidated yet. The invasiveness and metastasis of tumor cells are the result of degradation of connective tissue by matrix metalloproteinases (MMP). MMPs play a profound role on aggressiveness of tumor cells and their spread. This study was undertaken to understand the role of MMP genes and ß-catenin in gastric cancer biopsies and gastric cancer cell lines in relation to oxidative stress. Further, the aim of the study here was to determine the effect of MMP-3 dependent RNA interference on AGS cell line. The gene expression profiles of gastric cancer biopsies were investigated with RT-PCR, while the gene expression profiles of cell lines under stress conditions were determined through a Q-RT PCR study. The localization of ß-catenin and 8-OHdG in tissue samples and cell lines was determined by an immunohistochemical method. To determine MMP-3 expression level, MMP-3 siRNA was introduced into AGS cell line expressing MMP-3 protein and expression level was determined by a method utilizing Q-RT PCR. MMP and ß-catenin genes were determined to have a higher expression level in tumor tissues studied. Localization studies showed that, 85 % of ß-catenin was present in cytoplasm and membrane, while the rest was localized in nucleus. The toxicity of H2O2 on gastric cancer cell lines was similar to that of on normal cells but the response of cells to stress was different. One other striking finding was that MKN-45 cell line was determined to go to apoptosis upon treatment with H2O2, the other cell lines did not show such behavior. While the infection by H. pylori did not cause a change in the expression leveles of ß-catenin and MMP genes, H2O2 stress resulted an increased level of expression in MMP-1, MMP-7, MMP-14, MMP-15, MMP-17 and ß-catenin genes. Down-regulation of the expression of these genes under no H2O2 conditions showed that the difference in gene expression profiles was due to oxidative stress. The silencing of MMP-3 gene in AGS cell line caused a decrease MMP-15, but an increase in MMP-10 expression. It is assumed that, in gastric cancer the effect of MMP-3 expression has a key role on regulation of other MMPs.
Gastric cancer is the fourth leading cancer type of all cancers and although there has been a decline in the incidence of this cancer in recent years, it is still one of the most common cause of deaths. Although the role of oxidative stress on cancer formation has long been known, its mechanism has not been elucidated yet. The invasiveness and metastasis of tumor cells are the result of degradation of connective tissue by matrix metalloproteinases (MMP). MMPs play a profound role on aggressiveness of tumor cells and their spread. This study was undertaken to understand the role of MMP genes and ß-catenin in gastric cancer biopsies and gastric cancer cell lines in relation to oxidative stress. Further, the aim of the study here was to determine the effect of MMP-3 dependent RNA interference on AGS cell line. The gene expression profiles of gastric cancer biopsies were investigated with RT-PCR, while the gene expression profiles of cell lines under stress conditions were determined through a Q-RT PCR study. The localization of ß-catenin and 8-OHdG in tissue samples and cell lines was determined by an immunohistochemical method. To determine MMP-3 expression level, MMP-3 siRNA was introduced into AGS cell line expressing MMP-3 protein and expression level was determined by a method utilizing Q-RT PCR. MMP and ß-catenin genes were determined to have a higher expression level in tumor tissues studied. Localization studies showed that, 85 % of ß-catenin was present in cytoplasm and membrane, while the rest was localized in nucleus. The toxicity of H2O2 on gastric cancer cell lines was similar to that of on normal cells but the response of cells to stress was different. One other striking finding was that MKN-45 cell line was determined to go to apoptosis upon treatment with H2O2, the other cell lines did not show such behavior. While the infection by H. pylori did not cause a change in the expression leveles of ß-catenin and MMP genes, H2O2 stress resulted an increased level of expression in MMP-1, MMP-7, MMP-14, MMP-15, MMP-17 and ß-catenin genes. Down-regulation of the expression of these genes under no H2O2 conditions showed that the difference in gene expression profiles was due to oxidative stress. The silencing of MMP-3 gene in AGS cell line caused a decrease MMP-15, but an increase in MMP-10 expression. It is assumed that, in gastric cancer the effect of MMP-3 expression has a key role on regulation of other MMPs.
Açıklama
Anahtar Kelimeler
Biyoloji, Biology, Genetik, Genetics, Onkoloji, Oncology
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Gencer, S. (2009). Mide kanserinde matriks metalloproteinaz (MMP) genlerinin ifadesi üzerine oksidatif stres ve RNA müdahalesinin etkisi. Yayımlanmış Doktora lisans tezi, İnönü Üniversitesi, Malatya.
