Browsing by Subject "RNA-sequencing"
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(2020)Tiivistelmä – Referat – Abstract Telomeres are cap shaped structures at the very end part of each chromosome that protect DNA from degradation or unwanted chromosome-chromosome attachments. Telomere lengths show considerable heterogeneity in different cells of the same cell population. Reasons for heterogeneitiy and mechanisms inside cells causing them are not fully understood. In this study, we explored the correlation between telomere length and different gene expressions. First, using FACS technique we sorted each single cell into each well of 96-well plate. Second, we used SYBR green based qPCR for telomere length measurement. Third, we used Illumina-seq for sequencing extracted mRNAs. [6] We found a set of genes that were in strong correlation with telomere length, giving opportunity to explore the biological pathways. We compared pathways between different samples and found strong connections between genes involved in viral cycles and immune system with extracted genes that were in high correlation with telomere. We found heterogeneity of telomere lengths and transcriptomes in different cell lines. Telomere related proteins, specifically those involved in shelterin complex, are expressed highly in cancer cell lines and LPS-stimulated monocytes compared to the non-stimulated monocytes. In our study, SLC38A2, PURB, UBR3, SSR1, NCAPH2, AIMP2, PHF21A genes were highly correlated with telomere in mutual way and can therefore be considered as new biomarkers/novel candidates for telomere-related studies. The importance of these genes has been reported in aging/mortality. Concurrent with our findings, a recent report also suggested that NCAPH2 plays role in regulating telomere stability and maintenance through its interaction with TERF. [65] We found new genes in correlation with telomere regulation, and our findings are therefore of high importance in research of cancer, neurodegenerative diseases and aging. Further studies are, however, required as our data is limited by small number of samples and inability to properly validate our technique.
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(2019)Human umbilical vein endothelial cells are responsible for maintaining and forming new vessels from existing ones, in a biological process called sprouting angiogenesis. Sprouting angiogenesis is a crucial mechanism for the resolution of hypoxia and normal development of tissues. It also plays a key role in internal plague hemorrhages, which can lead to embolisms and other cardiovascular complications. Angiogenesis is also crucial for cancer development. Sprouting angiogenesis is initiated by hypoxic tissue excreted vascular endothelial growth factor gradient, which induces normal endothelial cells into either a proliferative stalk cell or a signal sensing tip cell phenotype. Both of these cell types depend on the rapid flow of lipids to their plasma membrane, either to form plasma membrane protrusions in tip cells or as new plasma membrane material in dividing stalk cells. This flow is envisioned to involve both vesicle-mediated and non-vesicular mechanisms. A major non-vesicular route of lipid transfer occurs at membrane contact sites via lipid transport proteins. Furthermore, lipids can be transported to the plasma membrane by the direct fusion of vesicles or endosomes with the plasma membrane This thesis set out to explore the role of two membrane contact site proteins, oxysterol-binding protein- related protein 2 and protrudin, in angiogenesis and lipid transfer. Their role was examined by RNA-sequencing transient knock-down samples of these proteins in HUVECs. The RNA-sequencing data was examined by differential expression, gene ontology overrepresentation and gene set enrichment analyses. Gene expression analysis provided almost 10 000 significantly changed transcripts (adjusted p-values < 0.05), in each silenced cell type. The distribution of differentially expressed genes in oxysterol-binding protein- related protein 2 silenced cells, is skewed toward negative fold changes, whereas the distribution of differentially expressed genes in protrudin silenced samples is normally distributed. The results also show significant changes in gene ontologies related to proliferation, cell cycle, angiogenesis as well as hypoxia in both sample types. Gene set enrichment analysis showed upregulation in angiogenesis related pathways, such as the PI3K-Akt and MAPK pathways, in both samples. Significant downregulation was present in cell cycle related pathways and cholesterol biosynthesis pathway in both ORP2 and protrudin silenced samples.
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