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Small Intestinal Neuroendocrine Tumor Analyses: Somatostatin Analog Effects and MicroRNA Profiling
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine oncology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets.

To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15.

To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells.

In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1039
Keyword [en]
Small intestinal neuroendocrine tumors, Somatostatin analogs, Laser-capture microdissection, Microarray profiling and microRNA profiling
National Category
Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-233207ISBN: 978-91-554-9058-4 (print)OAI: oai:DiVA.org:uu-233207DiVA: diva2:751006
Public defence
2014-11-20, Enghoffsalen, Entrance 50, Uppsala University Hospital, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-10-30 Created: 2014-09-30 Last updated: 2015-01-23
List of papers
1. The Somatostatin Analogue Octreotide Inhibits Growth of Small Intestine Neuroendocrine Tumour Cells
Open this publication in new window or tab >>The Somatostatin Analogue Octreotide Inhibits Growth of Small Intestine Neuroendocrine Tumour Cells
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, e48411- p.Article in journal (Refereed) Published
Abstract [en]

Octreotide is a widely used synthetic somatostatin analogue that significantly improves the management of neuroendocrine tumours (NETs). Octreotide acts through somatostatin receptors (SSTRs). However, the molecular mechanisms leading to successful disease control or symptom management, especially when SSTRs levels are low, are largely unknown. We provide novel insights into how octreotide controls NET cells. CNDT2.5 cells were treated from 1 day up to 16 months with octreotide and then were profiled using Affymetrix microarray analysis. Quantitative real-time PCR and western blot analyses were used to validate microarray profiling in silico data. WST-1 cell proliferation assay was applied to evaluate cell growth of CNDT2.5 cells in the presence or absence of 1 μM octreotide at different time points. Moreover, laser capture microdissected tumour cells and paraffin embedded tissue slides from SI-NETs at different stages of disease were used to identify transcriptional and translational expression. Microarrays analyses did not reveal relevant changes in SSTR expression levels. Unexpectedly, six novel genes were found to be upregulated by octreotide: annexin A1 (ANXA1), rho GTPase-activating protein 18 (ARHGAP18), epithelial membrane protein 1 (EMP1), growth/differentiation factor 15 (GDF15), TGF-beta type II receptor (TGFBR2) and tumour necrosis factor (ligand) superfamily member 15 (TNFSF15). Furthermore, these novel genes were expressed in tumour tissues at transcript and protein levels. We suggest that octreotide may use a potential novel framework to exert its beneficial effect as a drug and to convey its action on neuroendocrine cells. Thus, six novel genes may regulate cell growth and differentiation in normal and tumour neuroendocrine cells and have a role in a novel octreotide mechanism system.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-186023 (URN)10.1371/journal.pone.0048411 (DOI)000310600500127 ()
Available from: 2012-11-29 Created: 2012-11-27 Last updated: 2017-12-07Bibliographically approved
2. Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors.
Open this publication in new window or tab >>Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors.
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2013 (English)In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 26, no 5, 685-696 p.Article in journal (Refereed) Published
Abstract [en]

Well-differentiated small intestinal neuroendocrine tumors are rare malignancies. They arise from enterochromaffin cells and very little is known about differential microRNA (miRNA) expression. The aim of this study was to identify the miRNA profile of well-differentiated small intestinal neuroendocrine tumors, which may have a critical role in tumor development, progression and potentially develop miRNAs as novel clinical biomarkers. Specimens from two test groups, 24 small intestinal neuroendocrine tumor specimens at different stages of malignancy, are included in this study. Total RNA from the first test group, five primary tumors, five mesentery metastases and five liver metastases was hybridized onto the Affymetrix Genechip miRNA arrays to perform a genome-wide profile. The results were validated by using quantitative real-time PCR (QRT-PCR) and northern blot analyses. We then expanded the investigation to laser capture microdissected small intestinal neuroendocrine tumor cells and immuno-laser capture microdissected normal enterochromaffin cells of the first test group. Furthermore, a second test group, three primary tumors, three mesentery metastases and three liver metastases, was included in the study. Thus, two independent test groups validated the data by QRT-PCR. Moreover, we characterized nine miRNAs, five (miR-96, -182, -183, -196a and -200a), which are upregulated during tumor progression, whereas four (miR-31, -129-5p, -133a and -215) are downregulated. Several online software programs were used to predict potential miRNA target genes to map a number of putative target genes for the aberrantly regulated miRNAs, through an advanced and novel bioinformatics analysis. Our findings provide information about pivotal miRNAs, which may lead to further insights into tumorigenesis, progression mechanisms and novel therapeutic targets recognition.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-194041 (URN)10.1038/modpathol.2012.216 (DOI)000318456200008 ()23328977 (PubMedID)
Available from: 2013-02-07 Created: 2013-02-07 Last updated: 2017-12-06Bibliographically approved
3. Circulating microRNA detection in small intestinal neuroendocrine tumor patients treated with somatostatin analogs
Open this publication in new window or tab >>Circulating microRNA detection in small intestinal neuroendocrine tumor patients treated with somatostatin analogs
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2014 (English)Article in journal, Meeting abstract (Refereed) Submitted
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-233202 (URN)
Available from: 2014-09-30 Created: 2014-09-30 Last updated: 2015-01-23Bibliographically approved
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