Browsing by Subject "miRNA"
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Item BMPs Regulate the Oft Development via miRNAs(2014-08-04) Bai, YanCongenital heart diseases (CHD) are the leading cause of infant mortality and morbidity. Defects of the outflow tract (OFT) make up a large percentage of human CHD. In my study, I focused on the role of Bone morphogenetic protein (BMP) on heart development. I investigated Bmp signaling in OFT development using conditional knocking out Bmps, including Bmp2, -4 and -7. Deletion of Bmp4/7 in second heart field (SHF) results in persistent truncus arteriosus (PTA), which is caused by endothelial to mesenchyme transition (EMT) defect. I found that Vegfa (vascular endothelial growth factor a) is a downstream target of Bmp and miR-17-92. Repression of Vegfa in OFT is important for the proper onset of EMT at E10.5. Further exploration on Bmp2/4/7 mutation indicates that loss of Bmp signal disrupt the smooth muscle differentiation and maintenance. Similar with observations on Marfan syndrome mouse model, Bmp2/4/7 triple CKO embryos exhibited significant upregulation of Tgf-? signal activity. Alternation of SMC feature is likely due to the persistence of Isl-1 expression. Haploinsufficiency of Bmp4 triggered early onset of aorta aneurysm in Fbn-1^(C1039G/+) mice. These data indicate that Bmps not only play an important role in embryonic cardiogenesis, but also are critical for the cell fate differentiation and maintenance in adulthood.Item Characterizing miRNA mediated regulation of proliferation(2014-05) Polioudakis, Damon Constantine; Iyer, Vishwanath R.Cell proliferation is a fundamental biological process, and the ability of human cells to transition from a quiescent to proliferative state is essential for tissue homeostasis. Most cells in eukaryotic organisms are in a quiescent state, but on appropriate physiological or pathological stimuli, many types of somatic cells may exit quiescence, re-enter the cell cycle and begin to proliferate. The ability of cells to remain viable while quiescent, exit quiescence and re-enter into the cell cycle is the basis for varied physiological processes such as wound healing, lymphocyte activation and hepatocyte regeneration, but is also a hallmark of cancer. The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. Our understanding of miRNA biology has significantly improved, but the miRNA regulatory networks that govern cell proliferation are still largely unknown. We characterized a miR-22 Myc network that mediates proliferation through regulation of the interferon response and multiple cell cycle arrest genes. We identified several cell cycle arrest genes that regulate the effects of the tumor suppressor p53 as direct targets of miR-22, and discovered that miR-22 suppresses interferon gene expression. We go on to show that miR-22 is activated by the transcription factor Myc as quiescent cells enter proliferation, and that miR-22 represses the Myc transcriptional repressor MXD4, mediating a feed forward loop to elevate Myc expression levels. To more effectively determine miRNA targets, we utilized a combination of RNA-induced silencing complex immunoprecipitations and gene expression profiling. Using this approach for miR-191, we constructed an extensive transcriptome wide miR-191 target set. We show that miR-191 regulates proliferation, and targets multiple proto-oncogenes, including CDK9, NOTCH2, and RPS6KA3. Recent advances in determining miRNA targetomes have revealed widespread non-canonical miRNA-target pairing. We experimentally identified the transcriptome wide targets of miR-503, miR-103, and miR-494, and observed evidence of non-canonical target pairing for these miRNAs. We went on to confirm that miR-503 requires pairing outside of the canonical 5' seed region to directly target the oncogene DDHD2. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis.Item Computational identification and evolutionaty enalysis of metazoan micrornas(2009-05-15) Anzola Lagos, Juan ManuelMicroRNAs are a large family of 21-26 nucleotide non-coding RNAs with a role in the post-transcriptional regulation of gene expression. In recent years, microRNAs have been proposed to play a significant role in the expansion of organism complexity. MicroRNAs are expressed in a cell or tissue-specific manner during embryonic development, suggesting a role in cellular differentiation. For example, Let-7 is a metazoan microRNA that acts as developmental timer between larval stages in C. elegans. We conducted a comparative study that determined the distribution of microRNA families among metazoans, including the identification of new family members for several species. MicroRNA families appear to have evolved in bursts of evolution that correlate with the advent of major metazoan groups such as vertebrates, eutherians, primates and hominids. Most microRNA families identified in these organisms appeared with or after the advent of vertebrates. Only a few of them appear to be shared between vertebrates and invertebrates. The distribution of these microRNA families supports the idea that at least one whole genome duplication event (WGS) predates the advent of vertebrates. Gene ontology analyses of the genes these microRNA families regulate show enrichments for functions related to cell differentiation and morphogenesis. MicroRNA genes appear to be under great selective constraints. Identification of conserved regions by comparative genomics allows for the computational identification of microRNAs. We have identified and characterized ultraconserved regions between the genomes of the honey bee (Apis mellifera) and the parasitic wasp (Nasonia vitripennis), and developed a strategy for the identification of microRNAs based on regions of ultraconservation. Ultraconserved regions preferentially localize within introns and intergenic regions, and are enriched in functions related to neural development. Introns harboring ultraconserved elements appear to be under negative selection and under a level of constraint that is higher than in their exonic counterparts. This level of constraint suggests functional roles yet to be discovered and suggests that introns are major players in the regulation of biological processes. Our computational strategy was able to identify new microRNA genes shared between honey bee and wasp. We recovered 41 of 45 previously validated microRNAs for these organisms, and we identified several new ones. A significant fraction of these microRNA candidates are located in introns and intergenic regions and are organized in genomic clusters. Expression of 13 of these new candidates was verified by 454 sequencing.Item Determination of how miRNAs mediate repression in Drosophila and the essential role of the oskar mRNA in egg chamber development(2009-12) Reich, John Curtis; Macdonald, Paul M.; Fischer, Janice; Stein, David; Sullivan, Christopher; Gross, JeffreymiRNAs are important regulators of gene expression. These small RNAs function throughout development and regulate translation of a number mRNAs. miRNAs exert their affect on translation as part of the RNP complex RISC. RISC can affect translation of transcripts at both the level of translation initiation, and post-initiation. Although mechanisms of repression mediated by miRNAs have been intensively studied, repression is not well characterized. In order to understand how miRNAs regulate translation in Drosophila, we first characterized miRNA-mediated repression in the ovary. We developed an ovarian assay sensitive to regulation by miRNAs and found that regulated transcripts localize to cytoplasmic puncta distinct from sponge bodies, cytoplasmic RNP structures consisting of proteins implicated in miRNA-mediated regulation. In addition, we devised a genetic screen to identify genes involved in miRNA-mediated regulation. Seven mutants were isolated from the screen, and two mutants were subsequently mapped to separate 1Mb genomic regions. Both these regions are devoid of genes implicated in miRNA-mediated regulation, suggesting our mutants identify novel components involved in repression. The oskar mRNA encodes for the Oskar protein, which is vital in establishing the posterior axis of the Drosophila embryo. In addition to its protein coding function, the osk mRNA has another essential role: it is required for egg chamber progression through oogenesis. This role of oskar is mediated by its 3ʼ UTR, but how it functions in this role is unknown. Here, we investigate the function of the 3ʼ UTR and discover that the well-defined BRE sequences are required for egg chamber progression through oogenesis. The BREs mediate translational repression of the highly regulated oskar mRNA and were previously defined by their ability to bind Bruno, which represses translation of the oskar mRNA. We also provide evidence that the osk BREs sequester Bruno, potentially inhibiting Bruno from binding and misregulating other mRNAs. Our results suggest a novel regulatory loop, where oskar sequesters and inhibits Bruno from misregulating mRNAs, and Bruno, in turn, regulates translation of the oskar mRNA.Item Discovery and design of an optimal microRNA loop substrate(2013-05) Hwang, Tony Weiyang; Ellington, Andrew D.RNA interference, or RNAi, is a cellular mechanism that describes the sequence-specific post transcriptional gene silencing observed in plants, fungi, and metazoans, facilitated by short double-stranded RNAs and microRNAs (miRNAs) with sequence complementarity to target mRNAs. Many of the regulatory mechanisms of the RNAi pathway by which these small miRNAs are first processed, from primary transcripts to precursor miRNA stemloops and then to mature miRNAs, by the multiprotein complexes Drosha and Dicer, respectively, still remain unknown. Within the miRNA biogenesis pathway, there is strong evidence pointing to the terminal loop region as an important regulatory determinant of miRNA maturation. To further elucidate the terminal loop's exerted control over miRNA processing, we propose a combined in vitro / in vivo selection experiment of a randomized pri-miRNA terminal loop library in search of an optimally processed pre-miRNA substrate. Here, we report the isolation of a premiRNA terminal loop sequence that is favorably processed by Drosha in vivo but also functions as an effective cis-inhibitor of further pre-miRNA processing by downstream Dicer. This terminal loop also demonstrated modular properties of Dicer inhibition in two different miRNAs, and should prove useful in further elucidating the mechanisms of miRNA processing in context of a newly proposed Dicer cleavage model (Gu et al. 2012). In combination, these findings may have important implications in both Drosha and Dicer's direct role in gene expression and miRNA biogenesis, the regulatory proteins that modulate their respective functions, as well as the potential development of new design rules for the more efficient processing and targeting of miRNA-based technology and RNAi therapeutics.Item MicroRNA expression in canine mammary cancer(Texas A&M University, 2008-10-10) Boggs, Rene' MichelleMicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan? Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.Item MicroRNAs Function as Cis- and Trans- Acting Modulators of Clock Gene Expression in SCN and Peripheral Circadian Oscillators(2012-11-15) Shende, Vikram Ravindra 1982-The circadian system in mammals is arranged as a hierarchical network of oscillators, with the master pacemaker of circadian rhythms located in the suprachiasmatic nuclei (SCN) of the hypothalamus and peripheral oscillators in most other organ and tissue systems of the body. The molecular machinery responsible for generating circadian rhythms is composed of interlocked transcriptional-translational feedback loops with the gene Brain Muscle Arnt-like 1 (Bmal1) functioning as a core positive regulator. Using the mouse, Mus musculus as a model system, we studied the post-transcriptional mechanisms regulating Bmal1 expression in the SCN pacemaker and in peripheral oscillators. Target prediction algorithms were used to identify microRNAs (miRNAs) predicted to target Bmal1. We profiled the temporal expression of miR-142-3p in the mouse SCN in vivo and in an immortalized SCN cell line and observed robust circadian rhythms in its expression in the SCN. Following luciferase-reporter and site-directed mutagenesis analyses, we identified miR-142-3p as a bona-fide post-transcriptional repressor of Bmal1. The temporal expression of potential Bmal1-targeting miRNAs was also examined in the circulation in mouse serum. In mice housed in a light-dark cycle, diurnal oscillations were observed in serum levels of miR-152 and miR-494, but not miR-142-3p expression. Luciferase reporter studies indicated that miR-494, both independently and synergistically with miR-142-3p, repressed the Bmal1 3? UTR. Overexpression of these miRNAs disrupted ensemble circadian rhythms of PER2::LUCIFERASE activity in cultured fibroblasts. Overexpression of the miRNAs also increased their extracellular levels and their intracellular accumulation in recipient cultures exposed to conditioned medium. Furthermore, inhibition of exocytosis and endocytosis affected ensemble circadian rhythms in cultured fibroblasts. The results thus implicate miR-142-3p and miR-494 in the regulation of Bmal1 expression in the SCN and peripheral oscillators and suggest that miRNAs may function as both, intracellular and extracellular (cis- and trans- acting) signals, modulating the core clock mechanism in the SCN and in fine-tuning the synchronization of circadian rhythmicity between cell-autonomous oscillators in the periphery.Item Polyphenol-induced Anti-inflammatory and Cytotoxic Activities in Breast and Colon Cancer: Potential Role of miRNA's in Cell Survival and Inflammation(2013-12-11) Banerjee, NiveditaCancer is a major cause of death worldwide. Hence, there is a great need to develop novel therapeutic agents for the prevention and treatment of cancer in addition to conventional therapies. Dietary polyphenols are known to be effective in the prevention and treatment of several chronic diseases such as cancer, cardiovascular diseases, and diabetes. Particularly in carcinogenesis, polyphenols are known to suppress cancer growth, angiogenesis, and metastasis. Several studies have demonstrated that polyphenolics, ellagitannins, gallotannins, and chlorogenic acid from pomegranate, mango, and plum juice, respectively, are potent inhibitors of cancer cell proliferation and induce apoptosis and cell cycle arrest as well as decrease inflammation in vitro and vivo. The therapeutically relevant compounds in pomegranate are pelagic acid, ellagitannins, flavonoids, and 3-glucosides/3,5-diglucosides of the anthocyanins delphinidin, cyanidin, and pelargonidin that exerted antioxidant, anti-inflammatory, and anticarcinogenic activities in vitro and vivo. Mango pulp extract contains gallotannins, gallic acid, galloyl glycosides, and flavonoids such as quercetin and kaempferol glycosides, which showed antioxidant, anti-inflammatory, and anticarcinogenic activities in vitro and in vivo. Chlorogenic acid and neo-chlorogenic acid are contained in plum juice and are also known to function as chemoprevention and chemotherapeutic agents. The overall objective of this work was to investigate the underlying anti- inflammatory and cytotoxic mechanisms involving miR-27a-ZBTB10-Sp and miR-155- SHIP-1-PI3K axes, miR126-VCAM-1, miR126-PI3K/AKT-mTOR and miR143/PI3K/AKT/mTOR axes in polyphenol-mediated anti-inflammatory and anticarcinogenic activities in vitro and vivo. Pomegranate and Mango polyphenols exhibited antioxidant, anti-inflammatory, anticarcinogenic, and antiproliferative activities in vitro and in vivo. Polyphenols inhibited cell proliferation of breast cancer cell line BT474 and suppressed tumor growth in athymic BALB/c nude mice with BT474 xenografts. Interactions of Pg with miR-27a- ZBTB10-Sp and miR-155-SHIP-1-PI3K axes and mango miR126/PI3K/AKT axis were identified. In addition, pomegranate and plum polyphenols exerted cytotoxic and anti- inflammatory effects in azoxymethane AOM-treated rats and colon cancer cells. Interactions of Pg with miR126/VCAM-1 and miR126/PI3K/AKT/mTOR axes and plum with miR143/PI3K/AKT/mTOR were identified as mechanisms that at least in part appear to be involved in the anti-inflammatory and antiproliferative activities of pomegranate and plum polyphenolics. The presented research was conducted in order to understand the efficacy of polyphenols present in pomegranate, mango and plum and their underlying molecular mechanisms in different cancer models.Item Study of a tumor virus unveils a novel function for the miRNA biogenesis machinery(2012-12) Lin, Yao-Tang; Sullivan, Christopher S.Kaposi’s Sarcoma-associated Herpes Virus (KSHV) is a human herpesvirus associated with cancers. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. This work presented here is a novel approach to profile small RNAs from populations of cells undergoing predominantly lytic infection. Using two different next generation sequencing platforms, I cloned and sequenced both pre-microRNAs and derivative microRNAs (miRNAs). This analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5 prime end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. I report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miRs-K8 and K12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. I identify several novel small RNAs of low abundance, including viral microRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. This work also shows that much of the KSHV genome is transcribed in both the top and bottom strand orientations during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, I observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs). From the small RNA deep-sequencing, I also detected a low abundant small RNA fragment (23 nt) that maps to a putative hairpin structure (named hairpin K) within the KSHV PAN transcript. I demonstrate that hairpin K is a cis-negative regulatory element in PAN. It is well-appreciated that viruses utilize host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often utilize host-encoded factors to optimize replication. This work shows that Drosha, an endoribonuclease best known for its role in the biogenesis of miRNAs, can also function to directly regulate viral gene expression. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. I demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses.