Mikrorna-21 Baskılanmış Meme Kanseri Kök Hücrelerinin İnsan Kemik İliğinden İzole Edilen Telositler ve Telosit Kaynaklı Mitokondriler ile Ko-Kültürü

Abstract

The aim of this thesis study was to investigate the contribution of telocytes and telocyte-derived mitochondria, which have also been identified in breast tumours, to the tumour development of breast cancer stem cells via miR-21-5p. Within the scope of this thesis study, firstly, CD34 and CD117 positive telocytes were isolated from human bone marrow mononuclear cells and characterised by flow cytometry, immunofluorescent staining and TEM analysis methods and mitochondria were isolated from telocyte cells and characterised by immunofluorescent staining. In the next step, CD24- and CD44+ cancer stem cells were isolated from MDA-MB-231 breast cancer cell line and characterised by flow cytometry. Then, miR-21-5p expressions of cancer stem cells were suppressed by transfection of anti-miR-21-5p inhibitor. Changes in vimentin and ALDH1A1 protein levels were analysed by Western blot to investigate the role of bone marrow-derived telocytes and mitochondria in miR-21-5p suppressed cancer stem cell lines. SNAI1, LZTFL1, OCT3/4, ABCC11, CHD1 (E-Cadherin) and CHD2 (N-Cadherin) gene expression changes were analysed by RT-qPCR. Isolated telocytes expressed CD44/CD117/α-SMA/CD31/CD45 and CD34 surface markers and were also positive for vimentin and PDGF-β. Mitochondria isolated from telocytes were followed by immunofluorescent staining. Isolation and characterisation of cancer stem cells resulted in miR-21-5p inhibition and a 132-fold decrease in miR-21-5p gene expression was confirmed by RT-qPCR. After co-culture of telocytes and mitochondria with miR-21-5p inhibited cancer stem cells, miR-21-5p, SNAI1, ABCC11, N-Cadherin, vimentin and ALDH1A1 expressions decreased; E-Cadherin and LZTFL gene expressions increased. In this thesis, it has been demonstrated for the first time that telocytes and telocyte-derived mitochondria can reduce EMT transition via miR-21-5p in the progression of breast cancer and have the potential to reduce cancer metastasis by analyses at the gene/protein level.