Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous group of tumors that originate from various cell types belonging to the diffuse neuroendocrine system (DNES), which is ultimately derived from the endodermal primitive gut. The malignant transformations of these cells are caused by genetic alternation as for instance in the tumor suppressor MEN1 gene; however for most tumors the causation is not identified. GEP-NETs are generally well-differentiated with a benign course and can occur with typical syndromes due to hormone hypersecretion. But in most cases, the symptoms are nearly absent or vague that often results in delayed diagnosis (7-10 years), with an increased risk of developing metastasis. The most abundant type of GEP-NETs are carcinoids, which are neoplastic lesions derived from neuroendocrine cells of the gastrointestinal mucosa (55%); the remainder being pancreatic (PNETs), originating in most cases from the head (Insulinoma, Gastrinoma, Somatostatinoma and Non-functional PNETs) and tail (Glucagonoma and VIPoma) of the pancreas. Although these two subgroups differ in their histology and location, there are many diagnostic and therapeutic similarities between them. Today, clinical diagnostic is preferentially achieved by using serum/plasma CgA assay, advanced endoscopic and radiological imaging and due to their availability and routinely use, the diagnosis of benign and incidentally identified lesions have been increased over few decades, suggesting GEP-NETs not as rare as previously thought. Furthermore due to better treatments available, the survival of GEP-NET patients has been improved for the well-differentiated. The only potential curative solution for GEP-NETs is complete surgical resection. But as the majorities are metastatic lesions, more aggressive resection and embolization therapy are considered. The somatostatin-analogs, octreotide and lanreotide offer symptomatic relief and radiolabel somatostatin-analogs (PRRT) improve survival rate in most patients whereas conventional chemotherapy has only limited effect. Notch pathway is known to be involved in the control of cell fate choice between proliferation and differentiation in intestinal epithelial lineages and thus play an important role in both intestinal homeostasis and tumorigenesis. In most GEP-NETs investigated, Notch appears to be absent or minimal expressed, suggesting Notch to act as tumor suppressor. In thesis I examined the impact of miR-375 and miR-7-5p on the Notch mediator, RBP-Jκ in GEP-NETs with focus on its role in promoting tumorigenesis. Furthermore the prognostic value of miR-375 was considered in GEP-NET biopsy samples. First, miRNA expression profile of carcinoids was compared to normal tissue and it was observed that miR-375 and miR-7-5p was among the most upregulated. miR-375 and miR-7-5p are both identified as important for endocrine cells differentiation of islet pancreas. The miRNA expression profile was furthermore studied using laser capture microdissection and real-time RT–PCR analysis on ileal carcinoids were miRNA-375 expression in carcinoid tissue was increased compared to normal mucosal endocrine cells. In addition in situ hybridization in normal and tumor tissue samples showed that endocrine cells expressed miRNA-375 and miR-7-5p. Both in carcinoids from the upper and lower part of the intestinal tract and pancreatic islet tumors expressed miRNA-375 and miR-7-5p.To investigate the effect of miR-375 and miR-7-5p on GEP-NETs, NCI-H727 and CNDT2 cell lines was used as a model for carcinoids in vitro and cell proliferation was measured by xCELLigence system. In this thesis, knockdown of miR-375 in NCI-H727 induced cell proliferation compared to miR-375 and miR-7-5p overexpression. For CNDT2, cell proliferation only increased when miR-375 and miR-7-5p was overexpressed. Next, global analysis was used to predict miR-375 targets and the direct binding of miR-375 and miR-7-5p was studied using the 3‟-untranslated region (3‟UTR) of RBP-Jκ containing the binding sites, predicted by TargetScan. Luciferase reporter assay confirmed that miR-375 and miR-7-5p directly binds to RBP-Jκ 3‟UTR, however western blot analysis could not confirm that the protein is negatively regulated by the miRNAs in NCI-H727 cell line. In conclusion, miR-375 and miR-7-5p are upregulated in different GEP-NETs and are exclusively found in endocrine cells, which seem not to be dependent on the actual differentiation stage the cell are in. The specific location of the miR may be due to their importance for endocrine cell differentiation in tumors by negative regulating RBP-Jκ and thus keeping progenitor cells in differentiated state.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) KandidatMedicinalbiologi, (Bachelor/kandidatuddannelse) Kandidat|
|Udgivelsesdato||30 jun. 2015|
|Vejledere||Lennart Jr Friis-Hansen & Jesper Thorvald Troelsen|