Ovarian cancer results from the dysregulation, in normal ovarian epithelial cells, of genes responsible for the control of critical biological processes. Since their discovery 20 years ago, microRNAs have increasingly been implicated in that dysregulation due to their role mediating gene expression; changes in microRNA expression levels in cancer have been linked with tumor growth, proliferation and metastasis. Their imputed involvement in cancer has led to the possibility of their use as biomarkers and to their potential clinical use. Using mRNA and microRNA microarray analysis to compare human gene expression in normal ovarian surface epithelial (OSE) cells and epithelial ovarian cancer (EOC) cells, we explored the interactions between microRNAs and genes. First, we validated in silico predictions of microRNA targets by comparing them with in vitro evidence after exogenous microRNA transfection. We found that pairs of microRNAs with identical 7-nt (nucleotide) seed regions shared 88% of their predicted targets and 55% of their in vitro targets, confirming the importance of the seed as a targeting mechanism. But more importantly, we found that even a single nucleotide change in the seed region can result in a significant shift in the set of targeted genes, implying strong functional conservation of the seeds and their corresponding binding sites. Next, we discovered a 3-element network motif which explains the upregulation of nearly 800 genes in ovarian cancer which, as predicted microRNA targets, might be expected to be down- regulated. This model shows that, under certain circumstances, repressor genes which are down- regulated in cancer can apparently override the repressive effects of microRNAs, resulting in the upregulation of predicted microRNA targets. Finally, we developed a phenomenological network model, based on the Pearson correlation of microarray gene expression data, to identify subnetworks dysregulated in cell cycle and apoptosis. While our methodology reported many genes previously associated with ovarian cancer, it significantly suggested potentially oncogenic genes for further investigation. This network model can easily be extended to identify dysregulated genes in other cancers.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The solute carrier family 11 member 1 gene (SLC11A 1) is a divalent metal ion transporter with various pleiotropic effects on macrophage function. This gene that regulates iron, and is also regulated by cellular iron levels, has previously been linked to many infectious and autoimmune diseases. In this analysis, in vitro studies using the luciferase reporter system as well as case-control association studies were applied to investigate the significance of SLC11 A1 allelic variation in patients with diverse disease phenotypes. For in vitro studies, five different SLC11A 1 promoter constructs were generated, followed by transfection into U937 and THP-1 cells. The inserted fragments included two previously described alleles (alleles 2 and 3), two novel alleles identified in this study (alleles 8 and 9) and a C to T point mutation at nucleotide position -237 in the presence of allele 3. The most striking finding was the opposite effect observed for allele 3 in the presence of the -237C~ T polymorphism, similar to that of allele 2. Although the SLC11A 1 gene has previously been implicated in iron transport, we have demonstrated, for the first time, that the various alleles investigated cause differential expression of the gene upon iron loading. Association studies were performed by investigating diseases including oesophageal cancer (DC), inflammatory bowel disease (lBO) and hereditary haemochromatosis (HH) (or primary iron overload). Significant associations (P<O.05) were observed with allele 3 for all three conditions investigated only after stratification according to the presence of the -237C~ T polymorphism. Re-assessment of the promoter alleles according to expression profiles determined by the in vitro studies, showed statistically significant associations for allele 3 with DC and primary iron overload, compared with the respective population-matched control groups. Additionally, several novel variants were identified in exon 2 (112G~A, 148deIGACCAGCCC, 157insGACCAGCCCAG) and intron 1 (IVS1-28C~T), with variant IVS1-28C~T occurring at a significantly increased frequency in patients with DC compared with population-matched controls (P<O.05). Investigation of the SLC11A 1 gene in individuals presenting with iron overload in the absence of homozygosity for the HFE C282Y mutation, provided further support for the importance of sequence variation in the promoter region of the SLC11A 1 gene in modified risk of iron-related disorders. Genes related to iron homeostasis, including HFE, SLC11A3, HAMP and DCYTB, were investigated in individuals with similar criteria and potential disease-causing mutations were identified in 11% White and 45% Black South African patients. The possible significance of the SLC11A3 and DCYTB genes in iron overload in the Black South African population, and the possible involvement of the DCYTB gene in iron overload in general, are demonstrated for the first time. This study contributed to a better understanding of the function of the SLC11A 1 gene in relation to iron metabolism. The involvement of SLC11A 1 in a range of disease phenotypes including cancer and inflammatory conditions that may involve iron dysregulation, can probably be explained by interaction with external factors such as infectious agents that may affect cellular iron status. Our findings provide both in vivo and in vitro evidence that iron dysregulation mediated by allelic effects of SLC11A 1 may underlie disease susceptibility to infectious and autoimmune conditions. / AFRIKAANSE OPSOMMING: Die opgeloste stof draer familie 11 deel 1 geen (SLC11 A 1) is 'n divalente metaal ioon vervoerder met verskeie pleiotropiese effekte op makrofaagfunksie. Die geen, wat yster reguleer en ook deur sellulêre ystervlakke gereguleer word, is voorheen verbind met verskeie infektiewe en outo-immune siektes. In hierdie studie is in vitro analises, deur middel van die lusiferase verklikker sisteem, asook gevalle-kontrole assosiasie studies gebruik om die rol van SLC11A 1 alleel variasie in pasiënte met diverse siektefenotipes te ondersoek. Vyf verskillende SLC11A 1 promotor variante is geskep vir in vitro studies en gevolg deur transfeksie in U937 en THP-1 sellyne. Die ingevoegde fragmente het twee voorheen beskryfde allele (allele 2 en 3), twee nuwe allele wat in hierdie studie geïdentifiseer is (allele 8 en 9) en In C na T puntmutasie by nukleotied posisie -237 in die teenwoordigheid van alleel 3 ingesluit. Die opvallendste bevinding was die teenoorgestelde effek wat waargeneem is wanneer alleel 3 in die teenwoordigheid van die -237C~ T polimorfisme voorkom, soortgelyk aan alleel 2 uitdrukking. Alhoewel die SLC11A1 geen voorheen geïmpliseer is in yster vervoer, is daar vir die eerste keer aangetoon dat na yster lading, die verskillende allele differensiële uitdrukking van die geen veroorsaak. Verskeie siektes, insluitend slukderm kanker (OC), inflammatoriese dermsiekte (lBO) en oorerflike hemochromatose (HH) (of primêre ysteroorlading), is ondersoek deur middel van assosiasie studies. Betekenisvolle verskille (P<O.05) is waargeneem vir alleel 3 tussen die kontrole- en pasiëntgroepe in al drie siektes wat ondersoek is, maar slegs na stratifikasie volgens die teenwoordigheid van die -237C~ T polimorfisme. Na hersiening van die promotor allele volgens ekspressie profiele verkry met in vitro studies is statisties betekenisvolle assosiasie ook verkry vir alleel 3 met OC en primêre ysteroorlading in vergelyking met die onderskeie populasie kontrolegroepe. Verder is verskeie nuwe variante ook geïdentifiseer in ekson 2 (112G~A, 148deIGACCAGCCC, 157insGACCAGCCCAG) en intron 1 (IVS1- 28C~ T) en 'n statisties betekenisvolle verhoogde frekwensie van variant IVS1- 28C~ T is waargeneem in pasiënte met OC in vergelyking met die populasie kontrolegroep (P<O.05). Die belangrikheid van variasie in die promotor area van die SLC11A 1 geen as 'n modifiserende faktor in ysterverwante siektes, is verder ondersteun deur die SLC11A 1 geen in individue met ysteroorlading in die afwesigheid van homosigositeit vir die HFE C282Y mutasie te ondersoek. Ander gene geassosieerd met yster homeostase, insluitend HFE, SLC11A3, HAMP and DCYTB, is ondersoek in individue met soortgelyke seleksie kriteria en potensiële siekte-verwante mutasies is geïdentifiseer in 11% Wit en 45% Swart Suid-Afrikaanse pasiënte. Die moontlike belang van die SLC11A3 en DCYTB gene in ysteroorlading in die Swart Suid-Afrikaanse populasie en die moontlike betrokkenheid van die DCYTB geen in yster oorlading oor die algemeen, is vir die eerste keer aangetoon. Hierdie studie dra by tot 'n beter insig in die funksie van die SLC11A 1 geen ten opsigte van ystermetabolisme. Die betrokkenheid van SLC11A 1 in 'n reeks siekte fenotipes, wat insluit kanker en inflammatoriese toestande wat verband kan hou met 'n yster wanbalans, kan moontlik verklaar word deur interaksie met eksterne faktore soos infektiewe agente wat die sellulêre yster status kan beïnvloed. Ons bevindinge verskaf beide in vivo en in vitro getuienis dat yster wanbalans, wat bemiddel word deur alleliese effekte van SLC11A1, verantwoordelik mag wees vir vatbaarheid vir infektiewe en outoimmune siekte toestande.

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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

No description available.

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