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- All Subjects: microRNA
- Creators: LaBaer, Joshua
- Creators: Hale, Taben
MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.
I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
Reducing the amount of error and introduced data variability increases the accuracy of Western blot results. In this study, different methods of normalization for loading differences and data alignment were explored with respect to their impact on Western blot results. GAPDH was compared to the LI-COR Revert total protein stain as a loading control. The impact of normalizing data to a control condition, which is commonly done to align Western blot data distributed over several immunoblots, was also investigated. Specifically, this study addressed whether normalization to a small subset of distinct controls on each immunoblot increases pooled data variability compared to a larger set of controls. Protein expression data for NOX-2 and SOD-2 from a study investigating the protective role of the bradykinin type 1 receptor in angiotensin-II induced left ventricle remodeling were used to address these questions but are also discussed in the context of the original study. The comparison of GAPDH and Revert total protein stain as a loading control was done by assessing their correlation and comparing how they affected protein expression results. Additionally, the impact of treatment on GAPDH was investigated. To assess how normalization to different combinations of controls influences data variability, protein data were normalized to the average of 5 controls, the average of 2 controls, or an average vehicle and the results by treatment were compared. The results of this study demonstrated that GAPDH expression is not affected by angiotensin-II or bradykinin type 1 receptor antagonist R-954 and is a less sensitive loading control compared to Revert total protein stain. Normalization to the average of 5 controls tended to reduce pooled data variability compared to 2 controls. Lastly, the results of this study provided preliminary evidence that R-954 does not alter the expression of NOX-2 or SOD-2 to an expression profile that would be expected to explain the protection it confers against Ang-II induced left ventricle remodeling.
Premature babies are at risk of death from immature lung development. For this reason, pregnant mothers at risk for preterm delivery are administered dexamethasone (DEX), a synthetic glucocorticoid that promotes fetal lung development. However, exposure to DEX in utero is associated with low birth weight and cardiovascular development pathologies. Moreover, our lab found that DEX administration in-utero leads to a sex-specific increase in stress-induced tachycardia in female, but not male offspring. This project seeks to expand on this preliminary finding of the heart by examining local effectors of activity from the sympathetic system (tyrosine hydroxylase and catechol-o-methyltransferase). Tyrosine hydroxylase was measured as it catalyzes the rate limiting step of norepinephrine synthesis while catechol-O- methyltransferase was studied as it catalyzes the degradation of norepinephrine. Acetylcholinesterase was used to measure parasympathetic activity as it catalyzes the degradation of the primary neurotransmitter of the parasympathetic nervous system, acetylcholine. Analyses of sympathetic as well as parasympathetic activity were done to determine influences of in-utero DEX exposure on autonomic regulation in adulthood. Pregnant rats were administered DEX (0.4 mg/kg, i.p.) or vehicle (20% w/v 2-hydroxypropyl ß- cyclodextran) at gestation days 18-21, with euthanasia of offspring occurring at around the time the offspring reached 13-15 weeks of age. Left ventricles and right atria were pulverized, processed and subjected to western blot analysis to determine expression of proteins of interest. Males exposed to DEX in-utero saw a decrease in tyrosine hydroxylase expression in left ventricle and right atrium when compared to vehicle control, a difference not seen with females. In addition, catechol-o-methyltransferase expression was increased in right atria from male, but not female rats. Acetylcholinesterase expression was reduced in the right atria of female, but not male rats. The present findings suggest reduced norepinephrine signaling in the heart of male, but not female DEX-exposed offspring. Given that we have previously found that female, but not male rats exhibit exaggerated stress-induced tachycardia, our current findings suggest that males possess a sex-specific compensatory mechanism allowing the heart to resist increased sympathetic signaling from the brain, one that females do not possess. The underlying mechanics of this proposed mechanism are unclear, and further investigation is needed in this subject to determine the significance of the findings from our study.
The 5-year survival rate for late-stage metastatic melanoma is only ~30%. A major reason for this low survival rate is that one of the most commonly mutated genes in melanoma, NRAS, has no FDA-approved targeted therapies. Because the RAS protein does not have any targeted therapies, patients with RAS mutant tumors have an ongoing need for treatments that indirectly target RAS. This thesis project aims to identify expression and phosphorylation levels of proteins downstream of RAS in melanoma cell lines with the most common driver mutations. By analyzing the protein-level differences between these genetic mutants, we hope to identify additional indirect RAS protein targets for the treatment of NRAS mutant melanoma. RAS has several downstream effector proteins involved in oncogenic signaling pathways including FAK, Paxillin, AKT, and ERK. 5 melanoma cell lines (2 BRAF mutant, 2 NRAS mutant, and 1 designated wildtype) were analyzed using western bloting for FAK, Paxillin, AKT, and ERK phosphorylation and total expression levels. The results of western blot analysis showed that NRAS mutant cell lines had increased expression of phosphorylated Paxillin. Increased Paxillin phosphorylation corresponds to increased Paxillin binding at the FAT domain of FAK. Therefore, cell lines with increased FAK FAT – Paxillin interaction would be more sensitive to FAK FAT domain inhibition. The data presented provide an an explanation for the reduction in cell viability in NRAS mutant cell lines infected with Ad-FRNK. This information also has significant clinical relevance as researchers work to develop synthetic FAK FAT domain inhibitors, such as cyclic peptides. Additionally, cell lines with high levels of phosphorylated AKT showed a significant reduction in the amount of phosphorylated ERK. The identification of this inverse relationship may help to explain why BRAF and NRAS mutations are mutually exclusive. To conclude, NRAS mutant cell lines have increased expression of phosphorylated Paxillin and AKT which may explain why NRAS mutant cell lines are more sensitive to FAK FAT domain inhibition.