Baş-boyun skuamöz hücre karsinoması ve makrofaj hücrelerinin apelin/APJ ekseninde moleküler bağlantılarının araştırılması
Yükleniyor...
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kanser, kardiyovasküler hastalıklardan sonra dünyada en çok ölüm oranlarına sahip hastalıktır. Günümüzde kanser tedavisi kemoterapi, radyoterapi ve cerrahi müdahalelerle gerçekleştirilmektedir. Yakın zamanda bu tedavi girişimlerine ek olarak immünoterapötik yaklaşımlar da kullanılmaya başlanmıştır. Mevcut tedavilere ek olarak kullanılan immünoterapötik ajanlar tedavi yanıtlarında gözle görülür iyileşme sağlamıştır. Makrofajlar mononükleer sistemin ana hücreleridir. Sahip oldukları yüksek plastisite yetenekleri makrofajları birçok metabolik hastalığın patolojisinde etkin rol oynamasını sağlarken, yapılan çalışmalar kanser tedavisinde de kullanılabilecek bir alternatif olduğunu göstermiştir. Bu çalışmanın amacı; apelin peptididin makrofaj polarizasyonuna olan etkisinin araştırılmasıdır. Bu tez çalışmasında; RAW264.7 fare makrofaj hücreleri ile baş-boyun hücre karsinoması hücre hattı SCCL MT1 hücreleri kullanılmıştır. SCCL MT1 hücrelerinde apelin geni shRNA yöntemiyle baskılanmıştır. Apelin geni aktif ve bastırılan SCCL MT1 hücreleri RAW264.7 hücreleriyle ko-kültüre edilmiştir. Ko-kültürasyonun RAW264.7 hücrelerindeki inflamatuar genler üzerine etkisi ve apelinin baskılanmasının SCCL MT1 hücrelerindeki migrasyon ve invazyon ilişkili genler üzerine etkisi RT-PZR analizi ile belirlenmiştir. Anlamlı değişiklik gösteren genlere ait protein seviyeleri ELISA metodu ile değerlendirilmiştir. Apelinin baskılanması, SCCL MT1 hücrelerinden apelin ifadesinin azalmasına ve ko-kültüre edildiği makrofajlarda kontrole kıyasla pro-inflamatuar cevabın artmasına neden olmuştur. IL1β, IL6 ve TNFα gen ifadelerinde anlamlı artış görülmüştür. Anti-inflamatuar sitokin genlerinde ise IL10 gen ifadesi anlamlı olarak azalmıştır. Apelinin baskılanmadığı grupta ise pro-inflamatuar genlerde düşüş gözlenirken anti-inflamatuar genlerde artış görülmüştür. Bunların yanı sıra apelinin SCCL MT1 hücrelerinde baskılanması, kanser hücrelerinde migrasyon ve invazyon yeteneğini azaltmıştır. E-kaderin ifadesi artarken N-kaderin ifadesi anlamlı olarak azalmıştır. Ayrıca Twist, Slug, Snail ve CD44 ekspresyon seviyeleri de anlamlı olarak düşmüştür. Makrofaj polarizasyonunda etkili genler olan IL1β, IL6, IL10, TGFβ ve TNFα gen ifadelerindeki anlamlı değişimler nedeniyle ELISA yöntemi kullanılarak protein seviyeleri incelenmiştir. RT-PZR analizi verileriyle uyumlu sonuçlara ulaşılmıştır. Apelinin SCCL MT1 hücrelerinde baskılanması RAW264.7 hücrelerinde pro-inflamatuar cevabı arttırmıştır. Bu sonuçlara göre kanser hücrelerinden salınan apelinin makrofajların polarizasyonuna doğrudan etki ettiği görülmektedir. Elde edilen sonuçlar kanser mikroçevresinde M2 tip makrofajların yoğun olarak bulunmasının apelin peptidi ile ilişkili olabileceğini ve apelinin kanser hücrelerinde terapötik hedeflenmesinin makrofajların, kanser progresyonunun durdurulmasında rol oynayabileceğini düşündürmektedir. Ayrıca apelin kanser hücrelerinin migrasyon ve invazyon yeteneğini arttırarak agresif bir fenotip oluşmasını sağlamaktadır.
Cancer is the disease with the highest mortality rate in the world after cardiovascular diseases. Today, cancer treatment is carried out with chemotherapy, radiotherapy, and surgical interventions. Recently, immunotherapeutic approaches have been used along with these treatment interventions and have resulted in significant improvement in treatment responses. Macrophages are the main cells of the mononuclear system. While their high plasticity abilities enable macrophages to play an active role in the pathology of many metabolic diseases, studies have shown that they are an alternative that can also be used in cancer treatment. The aim of this study is to investigate the effect of apelin peptide on macrophage polarization. In this thesis study, mouse macrophage cell line RAW264.7 cells and head and neck cell carcinoma cell line SCCL MT1 cells were used. The apelin gene was suppressed by shRNA method in SCCL MT1 cells. Then, apelin gene activated and suppressed SCCL MT1 cells were co-cultured with RAW264.7 cells. The effect of co-culture on inflammatory genes in RAW264.7 cells and the effect of apelin suppression on migration and invasion-related genes in SCCL MT1 cells were determined by RT-PCR analysis. Finally, protein levels of genes with significant changes were calculated by ELISA method. Suppression of apelin reduced apelin expressed from SCCL MT1 cells and increased pro-inflammatory response in co-cultured macrophages compared to control. Significant increases were observed in IL1β, IL6 and TNFα gene expressions whereas anti-inflammatory cytokine genes, IL10 and TGFβ gene expressions were significantly decreased. However, in the treatment group where apelin was not suppressed, a decline was observed in pro-inflammatory genes, while an incline was observed in anti-inflammatory genes. In addition, suppression of apelin in SCCL MT1 cells decreased the migration and invasion ability of cancer cells. E-cadherin expression was increased while N-cadherin was significantly decreased. Moreover, Twist, Slug, Snail and CD44 gene expression levels were also significantly decreased. Due to the significant changes in the gene expression levels of IL1β, IL6, IL10, TGFβ, and TNFα which are effective in macrophage polarization, their protein levels were examined by ELISA method subsequently, and the results consistent with the RT-PCR analysis data were obtained. In conclusion, suppression of apelin in SCCL MT1 cells increased the pro-inflammatory response in RAW264.7 cells. According to these results presented here, it was found that apelin released from cancer cells directly affects the polarization of macrophages and the intense presence of M2 type macrophages in the cancer microenvironment may be related to the apelin peptide.. The therapeutic targeting of apelin in cancer cells may play a role in stopping the cancer progression of macrophages. In addition, apelin increases the migration and invasion ability of cancer cells and provides an aggressive phenotype.
Cancer is the disease with the highest mortality rate in the world after cardiovascular diseases. Today, cancer treatment is carried out with chemotherapy, radiotherapy, and surgical interventions. Recently, immunotherapeutic approaches have been used along with these treatment interventions and have resulted in significant improvement in treatment responses. Macrophages are the main cells of the mononuclear system. While their high plasticity abilities enable macrophages to play an active role in the pathology of many metabolic diseases, studies have shown that they are an alternative that can also be used in cancer treatment. The aim of this study is to investigate the effect of apelin peptide on macrophage polarization. In this thesis study, mouse macrophage cell line RAW264.7 cells and head and neck cell carcinoma cell line SCCL MT1 cells were used. The apelin gene was suppressed by shRNA method in SCCL MT1 cells. Then, apelin gene activated and suppressed SCCL MT1 cells were co-cultured with RAW264.7 cells. The effect of co-culture on inflammatory genes in RAW264.7 cells and the effect of apelin suppression on migration and invasion-related genes in SCCL MT1 cells were determined by RT-PCR analysis. Finally, protein levels of genes with significant changes were calculated by ELISA method. Suppression of apelin reduced apelin expressed from SCCL MT1 cells and increased pro-inflammatory response in co-cultured macrophages compared to control. Significant increases were observed in IL1β, IL6 and TNFα gene expressions whereas anti-inflammatory cytokine genes, IL10 and TGFβ gene expressions were significantly decreased. However, in the treatment group where apelin was not suppressed, a decline was observed in pro-inflammatory genes, while an incline was observed in anti-inflammatory genes. In addition, suppression of apelin in SCCL MT1 cells decreased the migration and invasion ability of cancer cells. E-cadherin expression was increased while N-cadherin was significantly decreased. Moreover, Twist, Slug, Snail and CD44 gene expression levels were also significantly decreased. Due to the significant changes in the gene expression levels of IL1β, IL6, IL10, TGFβ, and TNFα which are effective in macrophage polarization, their protein levels were examined by ELISA method subsequently, and the results consistent with the RT-PCR analysis data were obtained. In conclusion, suppression of apelin in SCCL MT1 cells increased the pro-inflammatory response in RAW264.7 cells. According to these results presented here, it was found that apelin released from cancer cells directly affects the polarization of macrophages and the intense presence of M2 type macrophages in the cancer microenvironment may be related to the apelin peptide.. The therapeutic targeting of apelin in cancer cells may play a role in stopping the cancer progression of macrophages. In addition, apelin increases the migration and invasion ability of cancer cells and provides an aggressive phenotype.
Açıklama
Doktora Tezi
Anahtar Kelimeler
Apelin, Baş-boyun, Kanser, Makrofaj, shRNA, Apelin, Cancer, Head and Neck, Macrophage
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Seçer Çelik, F. (2022). Baş-boyun skuamöz hücre karsinoması ve makrofaj hücrelerinin apelin/APJ ekseninde moleküler bağlantılarının araştırılması. (Yayımlanmamış doktora tezi). Necmettin Erbakan Üniversitesi, Sağlık Bilimleri Enstitüsü Tıbbi Biyoloji Anabilim Dalı, Konya
