TITLE of PART I: MICRORNA-205 IS A TUMOR SUPPRESSOR IN BREAST CANCER Major Professor: Dr. Yinyuan Mo MicroRNAs as a class of novel negative regulators have drawn intensive attention since their existence in mammals was discovered in 2001. As endogenous small non-coding RNAs, miRNAs are capable of silencing gene expression at the post-transcriptional level by translation blocking or/and mRNA degradation. MicroRNA has been reported to involve in diverse biological events. Although miRNA deregulation has been observed in many types of cancers including breast cancer, only few of these de-regulated miRNAs have been confirmed to be related with breast cancer progression. This study is to investigate the association of miRNAs with the development of breast cancer. In this study, we report that mir-205 is significantly under-expressed in breast tumors compared to the matched normal breast tissues. Similarly, breast cancer cell lines including MCF-7 and MDA-MB-231 express a much lower level of mir-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of mir-205 significantly inhibits cell proliferation and anchorage-independent growth as well as cell invasion. Furthermore, mir-205 was shown to suppress lung metastasis in a mouse model. Finally, western blot combined with the luciferase reporter assays demonstrated that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets of mir-205 and such mir-205-mediated suppression is likely through the direct interaction with the putative mir-205 binding site in the 3'-untranslated region (3-UTR) of ErbB3 and VEGF-A, suggesting that down-regulation of ErbB3 and VEGFA likely contributes to the suppression role of mir-205 in breast cancer cells. Together, these results suggest that mir-205 is a tumor suppressor in breast cancer. Given that mir-205 is downregulated in breast tumor specimens and breast cancer cell lines, it would be interesting to determine how mir-205 is regulated in cancer cells. Supprisingly, we found that agents which have been previously shown to induce other miRNAs, such as p53 induction, the phenolic compound kaempferol, hypoxia, and ROS stress have little effect on mir-205 expression, whereas de-methylation and UV treatment only a moderate effect on mir-205. Therefore, our studies indicate that mir-205 is a tumor suppressor in breast cancer progression, however, regulation of mir-205 still remains to be elucidated. Knowledge gained from these studies will provide more comprehensive understanding of how miRNAs are associated with tumor initiation and progression in breast cancer and how miRNAs are de-regulated during cancer development, and as a result, mir-205 may serve as a novel target for cancer therapy. TITLE of PART II: REGULATION OF ESTROGEN-INDEPENDENT GROWTH BY MICRORNAS Major Professor: Dr. Yinyuan Mo Tamoxifen (TAM) has proven to be effective in the treatment of breast cancer. The primary target of TAM in vivo is estrogen receptor (ER), and thus levels of ER expression are the best predictors of benefit from TAM. Unfortunately, over 30% of ER-positive tumors fail to respond to TAM therapy; moreover, those breast tumors that initially respond to TAM will frequently develop resistance to the treatment. Although it is believed that this is likely due to the activation of estrogen-independent signaling, the precise molecular mechanism is not fully understood. This poses a significant challenge to the determination of treatment options for ER positive breast tumors. In this study, we performed in vivo selection experiments using the breast cancer ER positive MCF-7 cells infected with a pooled human microRNA library. It is well known that MCF-7 cells do not grow tumors in a xenograft mouse model unless estrogen is provided. We found that the MCF-7 cells infected with the microRNA library, but not the vector control, did grow tumors, suggesting that certain microRNAs are able to support the estrogen-independent growth. To characterize these microRNA-induced tumors, we recovered cells from the tumors in cell culture and found that they are more resistant to TAM than the MCF-7 cells with vector control. Moreover, they are able to grow in estrogen-free medium. Further analysis revealed that these recovered cells are ER positive with highly activated pAkt. By genomic DNA based real-time PCR, we identified that 6 miRNAs have been enriched in the tumor genome. Co-infection of these 6 enriched miRNAs is able to confer MCF-7 cells estrogen-independent growth and tamoxifen resistance. Further analysis indicated that mir-101 alone is sufficient to induce in vitro estrogen-independent growth and tamoxifen resistance. Together, these results suggest that microRNAs are important players in regulating the estrogen-independent growth and thus, identifying such microRNAs and understanding the underlying mechanisms will provide new insight into estrogen-independent tumor growth and TAM resistance, and thus, these microRNAs may serve as novel targets for breast cancer therapy.

Macrophages are mononuclear phagocytic cells that have diverse roles within the body. Tissue specific macrophages, e.g. Kupffer cells, microglia and osteoclasts, have roles in tissue homeostasis, while circulating macrophages play an important role in the innate immune system. Macrophages detect the presence of pathogen associated molecular patterns (PAMPs) via a range of receptors known collectively as pathogen recognition receptors (PRRs). Detection of pathogens causes the macrophages to become ‘activated,’ during which the macrophages undergo extreme morphological and translational changes that enable the pathogen to be neutralised and other immune system components to be recruited. Macrophage activation must be carefully regulated and promptly resolved, as chronic inflammation is damaging to the host. MicroRNAs have emerged as one mechanism by which activation is regulated. MicroRNAs are small, non-coding pieces of RNA that function as a post-transcriptional regulatory mechanism. Their action is exerted through binding with a complementary region in the 3’ untranslated region (3’UTR) of the target mRNA. This binding, facilitated by the ribonuclear protein complex RISC, prevents successful translation of the mRNA into its protein product. MicroRNAs have been shown to function across species, throughout development and during the adult life-span. In the immune system, microRNAs are known to be required for correct formation of germinal centres and normal development of B- and T-cells. MicroRNAs have also been shown to be differentially regulated during macrophage activation with different stimuli. In particular, miR-155, miR-146a and miR-21 are associated with macrophage activation by lipopolysaccharide (LPS). The objective of this work was to further understand the role of microRNAs during macrophage activation with LPS. Two approaches were adopted. Firstly, the regulation of individual microRNAs in LPS-activated bone marrow derived macrophages (BMDMs) was characterised by the use of illumina small RNA sequencing. Secondly, the requirement of the global microRNA population during macrophage biology was investigated through the use of DGCR8 and Dicer knockout systems. In keeping with the large number of changes reported in mRNA translation upon activation, expression of >400 microRNAs were found to be differentially regulated by exposure to LPS. Twelve of these microRNAs were chosen for further study (miR- 142-3p, -146a, -15b, -155, -16, -191, -21, -27b, -30b, -322-5p, -378 and -7a). Individual knock-down of these microRNAs in the RAW264.7 macrophage-like cell line mostly demonstrated subtle, rather than dramatic changes to the activation marker genes studied by RT-QPCR analysis. However, knock-down of miR-146a, -15b, - 155 and -191 were able to significantly alter the expression of the activation marker genes (Tnf-a, Cox2, Cxcl2, Il-6 and Saa3). Interestingly, knock-down of miR-142-3p, miR-146a and miR-155 appeared to show cross-regulation of these microRNAs. The cell index (CI) data suggested that miR-191 and miR-21 influence adhesion in activated macrophages. Studies with the DGCR8 and Dicer knockout systems showed that the global microRNA population was required for successful differentiation of macrophages from embryonic stem cells, and for normal expression of differentiation and activation markers in bone marrow derived macrophages. Overall, these results show that dynamic expression of microRNAs is an integral part of the macrophage response to LPS.

Macrophages play a key role in maintaining the balance and efficiency of the immune response. TH2 cytokines IL-4 & IL-13, through shared IL-4Rα signalling, trigger a state of alternative activation in macrophages and also drive their proliferation. Alternatively activated macrophages (AAMΦ) are involved in the control of helminth infections and have also been implicated in tissue repair. However, TH2 weighted imbalance can result in inflammatory disorders such as asthma and fibrosis. Hence, macrophage responses must be tightly regulated. MicroRNAs, a short (~22nt) class of non-coding RNA, are one such immunomodulatory feedback mechanism that can regulate gene expression by targeting the 3’ UTR of mRNA resulting in destabilisation of the mRNA and/or inhibition of translation. With their ability for vast gene regulation, it was hypothesised that microRNAs could play a crucial role in the regulation of AAMΦ by targeting genes and pathways critical for their induction, maintenance & proliferation. Previously generated microarrays in the lab have allowed us to identify microRNAs differentially expressed in AAMΦ. In an effort to determine which microRNAs are genuinely associated with alternative activation, the first part of this project examined the expression profiles of ten shortlisted microRNA candidates under varying conditions of alternative activation, ranging from a reductionist in vitro IL-4/13 stimulation of macrophage cell lines to a complex in vivo TH2 mouse model of filarial infection. Profiling of microRNA expression under these conditions revealed that the expression of two IL-4Rα dependent microRNAs, namely miR-199b-5p and miR-378, along with another microRNA, miR-146, was highly regulated and consistently associated with alternative activation. The subsequent chapters of this thesis investigated the contribution of these microRNAs in regulating AAMΦ responses. Interestingly, we identified miR-199b-5p as being highly expressed in AAMΦ in vivo but not in vitro. Pathway analysis identified insulin signalling and other proliferative pathways such as PI3K/AKT as being highly targeted by miR-199b-5p. Overexpression of miR-199b-5p in RAW 264.7 cells resulted in a reduction in the rate of proliferation and a change in the levels of Insulin Receptor Substrate -1 (IRS-1), suggesting that miR-199b- 5p might regulate macrophage proliferation via insulin signalling. An alteration in the expression of YM-1 and RELM-α, markers characteristic of alternative activation, was also observed. MiR-199b-5p was successfully delivered to the lung and overexpressed in alternatively activated alveolar macrophages. No effect was observed on IL-4 induced proliferation, potentially due to the lack of significant insulin receptor and IRS-1 expression in alveolar macrophages. However, secreted levels of YM-1, but not RELM-α, were significantly reduced. MiR-378 is a microRNA that has previously been shown to be associated with AAMΦ through targeting of AKT-1; however, a direct influence of this microRNA on the regulation of this phenotype is yet to be determined. In this thesis, we have provided direct evidence of the impact of miR-378 deficiency on the regulation of AAMΦ and their responses using miR-378 KO mice. The ability of macrophages isolated from WT and KO animals to alternatively activate was studied in various systems both in vitro and in vivo. The influence of miR-378 deficiency on IL-4 induced proliferation was also addressed in vivo. Although the lack of miR-378 had no significant effect on IL-4 driven macrophage proliferation, results from this chapter support a role for miR-378 in the regulation of alternative activation through regulation of YM-1 and RELM-α expression. Lastly, to determine whether this regulation by miR-378 had functional consequences, we also utilised Litomosoides sigmodontis, a murine model of filarial infection. Due to experimental limitations, a concrete role for miR-378 in the context of infection could not be established. The final chapter of this thesis focuses on examining the role of miR-146 in the regulation of AAMΦ. MiR-146a is a highly studied microRNA that has previously been linked strongly to TH1 immune responses, especially classical activation of macrophages. However, a role for this microRNA in regulating AAMΦ is yet to be determined. Expression levels of miR-146a and miR-146b, the two isoforms of miR-146, were found to be differentially regulated upon alternative activation, with a decrease in miR-146a and increase in miR-146b expression in response to IL-4 both in vitro and in vivo. Based on this difference in expression and their known functions in suppressing excessive proinflammatory responses, it was hypothesised that miR-146a/b serve to regulate proinflammatory molecules (and signals) in a fine balance to allow efficient alternative activation to occur. However, the high sequence similarity between these two isoforms proved to be a hindrance to test this hypothesis in terms of shared targets. Therefore, the latter half of this chapter was devoted to the generation and optimisation of a stable cell line for the identification of microRNA targets using CLASH (cross-linking, ligation and sequencing of hybrids). In summary, the results from this thesis provide an important foundation for further studies of the functional role of microRNAs in the regulation of AAMΦ. Firstly, it characterises the expression profiles of ten different microRNAs differentially expressed during alternative activation. Secondly, for the first time, it identifies a role for miR-199b- 5p in the regulation of macrophage proliferation and activation. Thirdly, this thesis has provided direct evidence for the effect of miR-378 deficiency on AAMΦ responses. Lastly, it identifies and demonstrates the robust differential expression of two separate isoforms of the same microRNA (miR-146) under varying conditions of alternative activation, whose functional properties as regulators of the AAMΦ phenotype await further investigation.

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2014. / Submitted by Raquel Viana (raquelviana@bce.unb.br) on 2014-10-14T17:34:14Z No. of bitstreams: 1 2014_TulioCesardeLimaLins.pdf: 2695168 bytes, checksum: 84c7546416fcbd577356782924466469 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2014-10-15T17:20:59Z (GMT) No. of bitstreams: 1 2014_TulioCesardeLimaLins.pdf: 2695168 bytes, checksum: 84c7546416fcbd577356782924466469 (MD5) / Made available in DSpace on 2014-10-15T17:20:59Z (GMT). No. of bitstreams: 1 2014_TulioCesardeLimaLins.pdf: 2695168 bytes, checksum: 84c7546416fcbd577356782924466469 (MD5) / Variações no número de cópias (do inglês Copy Number Variation - CNV) são segmentos iguais a ou maiores que 1 kilobase que apresentam variações estruturais como deleções e duplicações. Constituem uma fração de variação genética importante, que interagem, modulam ou direcionam a expressão gênica. MicroRNA são pequenos RNA de aproximadamente 20 nucleotídeos que regulam a expressão gênica pós-transcricional. O objetivo desta Tese foi analisar a expressão de microRNA com relação às variações estruturais do número de cópias dos respectivos genes em humanos, as regiões de CNV-miRNA. Foram selecionadas 11 regiões de provável polimorfismo no número de cópias além de duas regiões invariáveis como controle positivo, que foram avaliadas por triagem molecular em PCR quantitativa em uma amostra de 103 indivíduos da população brasileira. Em seguida, uma segunda amostragem de 30 indivíduos tiveram DNA e RNA coletados de células mononucleadas do sangue (PBMC) e de epitélio bucal e RNA total do plasma para respectiva quantificação da expressão desses microRNA. Para elucidar questões relativas à dosagem gênica, a expressão de microRNA foi comparada com o número de cópias das respectivas regiões gênicas. Das regiões que apresentaram polimorfismo (CNV-miR-570, -miR-499, -miR-126, -miR-150, -miR-338, - miR-1275), apenas uma não teve o microRNA quantificado (CNV-miR-499) por não ter apresentado amplificação nos tecidos e também no plasma. Os microRNA miR-570 e miR-338, tiveram uma expressão relativa crescente de acordo com o número de cópias para frações de PBMC (e plasma somente para o miR-570), no entanto, estatisticamente não significativas. Numa simulação populacional pelo método Monte Carlo (n = 1.000), foi identificada diferença significativa para o miR-570, com taxa de mudança progressiva relativa entre ao número de cópias. A hipótese de que o número de cópias de uma região contendo genes miRNA pode afetar a regulação da expressão de genes abre uma perspectiva de novas pesquisas envolvendo a variabilidade fenotípica. ______________________________________________________________________________________________ ABSTRACT / Copy number variation (CNV) are DNA segments of 1 kilobase or larger, showing structural variations, such as deletions, insertions or duplications. It constitutes a significant fraction of genetic variation that directly interacts or modulates gene expression. MicroRNA is a class of small RNA of about 20 nucleotides that regulates post-transcriptional gene expression. The aim of this Thesis was to analyze the expression of microRNA regarding the number of copies of their respective genes in humans, the CNV-miRNA regions. Eleven regions of probable polymorphisms in addition to two invariable control regions were selected and evaluated by quantitative PCR molecular screening in a sample of 103 individuals of the Brazilian population. Then, a second sample of 30 subjects had DNA and RNA collected from blood mononuclear cells (PBMC), oral epithelium and total RNA from plasma, quantified the expression of those microRNAs and compared with the number of copies, aiming to elucidate the relative response to gene dosage. From the regions that showed polymorphism (CNV-miR-570, -miR-499, -miR-126, miR-150, -miR- 338, -miR-1275) only one was unable to have the microRNA quantified (miR-499) for failing to provide amplification in tissues and plasma. The microRNA miR-570 and miR-338 had an increased relative expression according to the respective number of copies for PBMC fraction (and plasma only for miR-570), though not statistically significant. In a population simulation by Monte Carlo method (n = 1,000), a significant difference was identified to miR-570, with progressive rate of relative change between the categories of number of copies. The hypothesis that the copy number variation of a region containing miRNA genes can affect the regulation of gene expression opens a new perspective for research regarding phenotypic variability.

Les métastases osseuses sont des complications fréquentes du cancer du sein, responsables sur le plan clinique d'hypercalcémie, fractures osseuses et douleurs, pour lesquelles, il n'existe que des traitements palliatifs. Les cellules tumorales de carcinomes mammaires qui métastasent au site osseux expriment des gènes qui favorisent le tropisme osseux de ces cellules ainsi que leur ancrage et développement dans la moelle osseuse. Les mécanismes moléculaires sous-jacents à ces processus sont contrôlés par l'expression génique des cellules tumorales qui interagissent avec le microenvironnement et les cellules osseuses. Dans ce contexte, les microARNs en tant que régulateur endogène de l'expression génique, interfèrent avec les différentes étapes de la formation des métastases osseuses, incluant l'échappement des cellules tumorales de la tumeur primaire, la dissémination et l'invasion du site osseux, ainsi que l'apparition de lésions ostéolytiques. Les profils transcriptomiques des microARNs de cellules tumorales mammaires à caractère ostéotropique montrent que l'expression de la famille de microARNs-30s (miRs-30) est inhibée dans ces cellules. En clinique, la faible expression des miRs-30 est associée à un mauvais diagnostique de rechute et au statut hormono-résistant. Dans un modèle animal de métastases osseuses, l'expression forcée des miRs-30 dans des cellules tumorales qui métastasent fortement et spécifiquement à l'os, inhibe la formation des métastases osseuses. Nous montrons que les miRs-30 inhibent l'invasion et stimulent l'ostéoblastogenèse, in vitro et réduisent la charge tumorale et l'ostéoclastogenèse, in vivo. En accord avec ces résultats, l'expression de gènes qui stimulent les métastases osseuses est inhibée par les miRs-30. Parmi ces gènes, l'expression du CTGF (connective tissue growth factor) est augmentée dans les métastases osseuses humaines. Les étapes précoces des métastases osseuses sont étudiées par inoculation de cellules tumorales métastatiques murines dans la glande mammaire de souris. Dans ce modèle, les miRs-30 n'altèrent pas la croissance tumorale et la dissémination métastatique à l'os. Cependant, les miRs-30 inhibent l'invasion et le caractère de cellules souches tumorales de ces cellules métastatiques. Ces résultats suggèrent que les miRs-30, en régulant négativement les métastases osseuses, représentent une thérapie potentielle pour réprimer des gènes cibles qui stimulent les métastases osseuses / Bone metastasis is a common complication of advanced breast cancers and is clinically responsible of bone fractures, hypercalcemia and pain for which only palliative therapies are proposed. Breast tumor cells that preferentially invade bone express a set of deregulated genes that enhance bone tropism and facilitate bone marrow engraftment which may lead to the formation of overt osteolytic lesions. Molecular pathways underlining these steps are regulated through the tight control of genes expressed by cancer cells interacting with cells from the bone microenvironment. In this context, microRNAs act as regulators of gene expression and control multiple aspects of bone metastasis, including tumor cell escape from the primary site, dissemination, invasion of the bone marrow and secondary outgrowth. MicroRNA transcriptomic profiling of osteotropic breast cancer cell lines identified drastic down-regulation of the miR-30 family (miRs-30). In the clinic, low expression of miRs-30 in breast primary tumors is associated with poor distant metastasis-free survival and hormoneinsensitive status. In a model of human bone metastasis in vivo, the forced expression of miRs-30 in a breast cancer cell line that is highly and specifically metastatic to bone inhibited bone metastasis. We demonstrated that miRs-30 inhibit tumor cell invasiveness and stimulate osteoblastogenesis, in vitro, and reduces tumor burden and osteoclast activity, in vivo. Consistent with that, the expression of several genes that promote bone metastasis were inhibited by miRs- 30. Among these, expression of connective tissue growth factor (CTGF) was up-regulated in human bone metastasis. The early steps of bone metastasis were studied in a mouse model using spontaneously metastatic mouse breast cancer cell lines inoculated in the mammary gland. In this model, miRs-30 did not alter tumor growth or metastatic dissemination to bone. However, miRs-30 inhibited cell invasiveness and cancer stem cell-like phenotype of these metastatic cells. We conclude that miRs-30, by interfering negatively with bone metastasis, represent a potential therapy to repress gene targets that promote bone metastasis

Repetitive head impacts (RHI) and traumatic brain injuries are risk factors for the neurodegenerative diseases chronic traumatic encephalopathy (CTE) and amyotrophic lateral sclerosis (ALS). Although distinct, these diseases can share an overlapping pathology (e.g. TDP-43) and affect similar brain regions. However, the pathways involved and biomarkers for diagnosis are unknown. MicroRNAs (miRNAs) are altered in disease, are involved in gene regulation, and may be useful as stable biomarkers. Thus, we set out to determine associations between miRNA levels and disease state within the prefrontal cortex in a group of deceased participants with no pathology (controls), CTE, ALS, or comorbid CTE+ALS. Of the 47 miRNAs previously implicated in neurological disease, 27 were significantly different between pathology groups. Of these, seventeen (63%) were upregulated in both ALS and CTE and included miRNAs involved in inflammatory, apoptotic and cell growth/differentiation pathways. Nine miRNAs (33%) were specifically upregulated only in ALS as opposed to only one miRNA (4%) with CTE specific upregulation. Surprisingly, few miRNAs (14%) were significantly altered in comorbid CTE+ALS, which may reflect the milder disease present at death in participants with both conditions. Overall, we found patterns of miRNA expression that are common and unique to CTE and ALS and that suggest common pathways of pathogenesis.

miRNAs 29b and 181a down-regulate expression of the norepinephrine transporter in PC12 cells. M.X. Deng, G. A. Ordway and M.-Y. Zhu. Dept. of Biomedical Sciences, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The noradrenaline transporter (NET) is a noradrenergic marker and regulates neurotransmitter signaling by rapidly clearing released norepinephrine from synapses. Our previous studies demonstrated that rat NET mRNA and protein levels are regulated by chronic stress and by administration of corticosterone. Whether miRNAs are intermediaries in the regulation of NET expression remains to be elucidated. The present study was undertaken to determine possible regulatory effects of miRNAs on NET expression in PC12 cells, a cell model for noradrenergic neurons. Using computational target prediction, we identified several miRNAs potentially related to regulation of NET expression. Mimics of these miRNAs were transfected into PC12 cells. NET protein expression was assayed by Western blotting 48 hours after transfection. miR29b- and miR181a-transfected cells showed significantly reduced NET protein levels. To identify the exact target loci, the 3’-UTR of NET mRNA was amplified by PCR from PC12 genomic DNA and cloned downstream of the red firefly gene of the pmirGlo vector. The NET 3’-UTR-bearing pmirGlo and miR29b or miR181a were co-transfected into PC12 cells and luciferase signals were measured 48 hours after transfection. Consistent with Western blots, co-transfection of these miRNAs with rat NET3’-UTR-containing plasmids resulted in reduced levels of luciferase activity in PC12 cells. We conclude that miR29b and miR181a can function as negative regulators of NET translation in vitro. Further studies to determine whether these miRNAs contribute to the regulation of NET expression induced by antidepressants are under way.

No description available.

No description available.

Follicular dendritic cells (FDCs) are key elements of secondary lymphoid organs where they form the stromal component of B-cell follicles. FDCs possess extensive dendritic process that trap intact antigen via Fc and complement receptors on the cell surface. The antigen is displayed to B-cells, providing a basis for selection of high affinity B cells. FDC also have important roles in facilitating the clearance of apoptotic B cells by the secretion of the opsonising factor MFGE8. It is well established that lymphotoxin signalling is required for FDC maturation but the specific details of the molecular mechanisms that regulate FDC development and differentiation are not fully understood. MicroRNAs (miRNAs) are non-coding RNAs of approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level. MiRNAs bind to their target gene transcripts as part of the RNA induced silencing complex and repress translation of the target gene product. The objective of this study was to identify miRNAs that play a role in the development and function of FDCs. An in vivo murine model of FDC de-differentiation was used to provide material for miRNA analysis. By comparison of miRNA profiles from spleen tissue with FDC at different stages of de-differentiation, we would be able to obtain a miRNA signature for mature FDC. Spleens were collected at various time points over a 28 day period following transient blockade of lymphotoxin signalling. A variety of methods were used to profile the miRNAs expressed at different time points during the suppression and recovery of the FDC network. Comparison of the miRNA profiles of spleens containing mature, partially de-differentiated, and fully de-differentiated FDC identified a number of miRNAs that were differentially expressed during FDC de-differentiation. To assess the role of specific miRNAs in FDC development, the mouse FDC-like cell line, FL-YB, was used as an in vitro model system. FL-YB cells were used to perform gain-offunction and loss-of-function studies on selected miRNAs and to assess the effects of various stimuli/conditions on miRNA expression. The effects of different treatments on cell proliferation, morphology and adhesion, and on gene expression by FL-YB, were monitored. Loss-of-function studies for one of the selected miRNA (miR-100-5p) revealed a significant effect on a number of gene transcripts involved in mediation of the germinal centre response (Il-6, Tlr4, Ptgs1/2). These data indicate that miR-100-5p has a role in regulating Il-6, Tlr4 and Ptgs1/2 transcripts. None of these transcripts contain predicted target sites for miR-100-5p and so the effect of miR-100-5p on these transcripts is likely to be indirect. Further studies on these miRNA: target interactions are required to elucidate the mechanisms and biological consequences of miRNA regulation in FDCs.