Browsing by Subject "MicroRNAs"
Now showing 1 - 20 of 32
- Results Per Page
- Sort Options
Item Antagonistic Roles of miR-199a-3p/miR-214 and the miR-200 Family in the Regulation of Uterine Contractility During Pregnancy and Labor(2014-02-03) Williams, Koriand'r; Hammer, Robert; Mendelson, Carole R.; Mahendroo, Mala; Olson, Eric N.Progesterone (P4) and estradiol-17β (E2) play critical and opposing roles in regulating myometrial quiescence and contractility during pregnancy and labor (Kamel et al., 2010). While these contrasting hormonal effects are likely mediated via differential regulation of inflammatory and contractile genes, the underlying mechanisms remain incompletely understood. Recently, we discovered that miR-200 family members, miR-200b and miR-429, and their target, transcription factor ZEB1, serve as P4/progesterone receptor (PR)-mediated regulators of uterine quiescence during pregnancy (Renthal et al., 2010). In the present study, we identified a novel role for another miR-200 family member, miR-200a, to enhance local metabolism of P4 in myometrium and, thus, decrease PR function during the progression towards labor (Williams et. al., 2012a). This occurs via miR-200a repression of signal transducer and activator of transcription (STAT)5b, a transcriptional repressor of the P4-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD). We observed that miR-200a expression increased and STAT5b expression coordinately decreased in myometrium of mice as they progressed to labor and in laboring myometrium from pregnant women. These changes were associated with a dramatic increase in expression and activity of 20α-HSD in laboring myometrium from mouse and human. In a progesterone-withdrawal mouse model of preterm labor, preterm labor was associated with increased miR-200a, decreased STAT5b and enhanced 20α-HSD expression. In other studies, we also found that levels of the clustered miRNAs, miR-199a-3p and miR-214, were significantly decreased in laboring myometrium of pregnant mice and humans and in a inflammatory mouse model of preterm labor, while the miR-199a-3p/miR-214 target, cyclooxygenase-2 (COX-2), a critical enzyme in synthesis of pro-inflammatory prostaglandins, was coordinately increased (Williams et al., 2012b). The physiological relevance of the labor-associated increase in miR-199a-3p/214 expression was highlighted by the finding that overexpression of miR-199a-3p and miR-214 in cultured human myometrial cells inhibited COX-2 protein and blocked TNF-α-induced myometrial cell contractility. Notably, estrogen and P4 treatment of ovariectomized mice have opposing effects on uterine miR-199a-3p/214 expression that were mediated by ZEB1. Whereas, P4 stimulated ZEB1 and upregulated miR-199a/214 expression in mouse and human myometrium (Renthal et al., 2010), estrogen had an opposing inhibitory effect. Notably, ZEB1/2 inhibit miR-200 family expression. Together, our findings point to the key pivotal roles of myometrial ZEB1 and its miRNA targets as a hormonally-controlled regulators of inflammatory and contractile gene expression in the pregnant uterus during term and preterm labor.Item Characterization of Non-Coding RNAs in Regulating Thymic Epithelial Cell Responses to Pathophysiological Stress(2016-07-26) Hoover, Ashley Renae; Hooper, Lora V.; van Oers, Nicolai S. C.; Cleaver, Ondine; Mendell, Joshua T.The thymus is uniquely sensitive to several forms of stress. Stress initiates a transient involution that can reduce overall thymic volume up to 90%. The thymus is predominantly composed of developing thymocytes and specialized epithelial cells. The type of stress predicates whether the thymocytes or epithelial cells initiate the involutionary response. MicroRNAs (miRs) are small non-coding RNAs ~18-22 nucleotides in length that maintain cellular homeostasis and regulate stress responses. Previous work in the laboratory identified several microRNAs involved in regulating thymocyte responses to stress. Thymocytes have been the main population studied in response to stress. However, it has become increasingly clear that the epithelial cells play a critical role in thymus involution and the subsequent recovery of thymopoiesis. The work presented in this thesis characterizes an epithelial specific miR, miR-205, and its surrounding long noncoding RNA, MIR205.001 in regulating TEC functions. A deficiency of miR-205 specifically in TECs renders these cells more susceptible to stress mediated thymic involution. This is revealed by a significant loss in developing thymocytes, altered migration, delayed recovery of single positive thymocyte selection, and proliferative defects in cortical TECs. Gene expression comparisons revealed miR-205 deficient TECs had reduced levels of the TEC master transcriptional regulator, Foxn1, as well as the expression of multiple chemokines. MiR-205 mimics introduced into miR-205 deficient fetal thymic organ cultures were able to restore the levels of Foxn1 and selected chemokines. This work demonstrates that miR-205 positively regulates Foxn1 and chemokine expression following stress. MiR-205 resides within a putative long noncoding RNA (lncRNA), MIR205.001. TECs deficient in this lncRNA are also sensitive to stress, but do not experience a delay in thymopoiesis nor cortical TEC proliferation defects. This suggests these noncoding RNAs have non-overlapping functions within the TECs. Mice deficient in MIR205.001 also have distinct phenotypes, displaying reduced mendelian ratios indicative of a lethality. The surviving animals display reduced body size, weight, and fat mass. Current experiments are addressing whether this is a metabolic defect or due to changes in feeding behavior.Item Characterization of Stress Responsive MicroRNAs and Their Roles in T Cell Development(2014-01-06) Belkaya, Serkan; Hooper, Lora V.; van Oers, Nicolai S. C.; Chen, Zhijian J.; Yarovinsky, Felix; Liou, JenPhysiological stress evokes rapid changes in both the innate and adaptive immune responses. Immature αβ T cells developing in the thymus are particularly sensitive to stress, with infections and/or exposure to lipopolysaccharide or glucocorticoids eliciting a rapid apoptotic program. MicroRNAs (miRs) are short, non-coding RNAs that play critical roles in the immune system by targeting diverse mRNAs. We hypothesized that a subset of thymically encoded miRs would be stress responsive and modulate thymopoiesis. Thymic miR profiling revealed 18 distinct miRs that are dysregulated more than 1.5-fold in response to lipopolysaccharide or the synthetic glucocorticoid dexamethasone. These stress-responsive miRs are dynamically regulated in distinct thymocyte subsets. We utilized both transgenic and gene-targeting approaches to study the impact of these miRs on thymopoiesis under normal and stress conditions. MiR-181d is the most down-regulated thymic miR in response to stress. The over-expression of miR-181d in developing thymocytes reduced the number of immature CD4+CD8+ thymocytes. Lipopolysaccharide or dexamethasone injections caused a 4-fold greater loss of these cells than in the wild type controls. The targeted elimination of miR-181d resulted in a thymus stress-responsiveness similar to wild-type mice, suggesting a functional redundancy between miR-181 family members. Gene expression comparisons further indicated that miR-181d affects a number of stress, metabolic, and signaling pathways. These findings demonstrate that selected miRs enhance stress-mediated thymic involution in vivo. MiR-185, another stress-responsive miR in murine thymus, is haploinsufficient in almost all individuals with 22q11.2 deletion/DiGeorge syndrome that can present with immune, cardiac, parathyroid, and psychological problems. The molecular targets of miR-185 in thymocytes are unknown. Transgenic expression of miR-185 attenuated thymopoiesis at the TCRβ-selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, NFATc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, while a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with the haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion/DiGeorge syndrome patients. These findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.Item Decreased microRNA-122 Levels with HCV Clearance in HIV-HCV Co-Infections(2013-01-22) Dubin, Perry H.; Yuan, Hejun; Devine, Robert K.; Jain, Mamta K.; Hynan, Kinda S.; Lee, William M.BACKGROUND AND AIMS: Micro RNA-122 (miR-122) is under investigation as a target for direct antiviral agents against the hepatitis C virus (HCV), and as a biomarker for both cancer and acute liver injury. Previous data suggest HCV mono-infection is associated with increased serum miR-122 levels. This study sought to determine outcomes in regard to miR-122 levels following clearance of HCV in human immunodeficiency virus (HIV) co-infected patients. METHODS: Nine HCV-HIV co-infected patients undergoing antiviral therapy were treated with interferon and ribavirin for 48 weeks between January 2009 and March 2011, and had serial miR-122 levels measured in triplicate from serum with mirVanaTM PARISTM kit according to the instructions from the manufacturer (Ambion, AM1556). Values were measured at baseline, 1 week, 4 weeks, end of treatment (EOT; 48 weeks), and at 24 weeks after treatment completion (SVR24). SAS V9.3 was used to analyze these data. Change from baseline (copies/μL) was calculated as Log10 (Baseline)-Log 10(time), where time was 1 week, 4weeks, EOT, and SVR24; a repeated measures ANOVA was used to compare the results over time for the patients. If the ANOVA was found significant, post hoc, pairwise comparisons were used to examine change from baseline across the four time points. RESULTS: Six of nine achieved SVR24, 1 was undetectable at EOT but relapsed, and 2 patients were non-responders. Among the 6 patients achieving SVR, all showed a decrease in miR-122 levels between 0.16 and 1.46 logs, between baseline and SVR24. The ANOVA confirmed a significant decrease in miR-122 levels from 1 week to SVR24 (p=0.0225). Significant pairwise comparisons for change from baseline were found at 1 week versus SVR24 (p=0.0063), 4 weeks versus SVR24 (p=0.0086), and EOT versus SVR24 (p=0.0458). CONCLUSION: Clearance of chronic HCV is associated with decreased miR-122 levels in HIV co-infected patients and was not improved in patients with continued infection who failed to respond to treatment.Item The Functional Roles of the Lin28/let-7 Axis in Tissue Regeneration and Cancer(2017-04-11) Nguyen, Liem Hieu; Corey, David R.; Mendell, Joshua T.; Castrillon, Diego H.; Zhu, HaoThe let-7 microRNAs and their antagonists, the Lin28 RNA-binding proteins (Lin28a and Lin28b), are post-transcriptional regulators well known for their initially discovered role in controlling developmental timing, or heterochrony. Subsequent studies have further uncovered many additional cellular processes, such as differentiation, metabolism, and body size, controlled by Lin28 and let-7. Their role in pathology, however, is not as well characterized. Although several lines of in vitro evidence have implicated involvement of Lin28a/b and let-7 in cancer, their functional roles in tumor initiation and maintenance have not been vigorously demonstrated in animal models. The work in this thesis focuses on defining the in vivo functional roles of Lin28 and let-7 in the context of tissue regeneration and liver cancer. Our first study led us to discover that Lin28 overexpression is both sufficient to initiate liver cancer and necessary for its maintenance and that its oncogenic effects are mediated in part through downregulating let-7 and upregulating the Igf2-binding protein family expression levels. Subsequently, we demonstrated that overexpression of let-7g has a potent tumor suppressing effect at the expense of efficient tissue regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Surprisingly, we found that loss of only two let-7 members, let-7b and let-7c2, render an increased regenerative capacity in liver. Lastly, in an effort to translate our findings for clinical applications, we collaborated with Daniel Siegwart's group to engineer novel lipid nanoparticles that enabled us to efficiently deliver small RNAs, such as let-7, into mice and investigate their therapeutic effects. Together, this work carefully defined the functional role of each of the main players of the Lin28/let-7 pathway in cancer. This knowledge has clinical implications because it identifies new targets for drug developments to treat liver cancer, disease that currently has very limited treatment options.Item HP1BP3, A Chromatin Retention Factor for Co-Transcriptional MicroRNA Processing(2016-06-27) Liu, Haoming; Tu, Benjamin; Liu, Qinghua; Roth, Michael G.; Orth, KimRNA interference (RNAi) is a post-transcriptional gene silencing mechanism found in all eukaryotic organisms. It is characterized by a family of small non-coding RNAs, either endogenous (in the case of microRNAs) or exogenous (in the case of siRNAs), that inhibits gene expression post-transcriptionally. MicroRNAs (miRNAs) are a family of ~21-nt cellular RNAs that govern numerous pathological and physiological processes by mediating translational repression and deadenylation/decay of cognate mRNA. Dysregulation of miRNA expression have been associated with various types of cancer and developmental diseases. Typically, primary (pri-)miRNA transcripts are processed by Drosha complex into precursor (pre-)miRNAs, and then by cytoplasmic Dicer complex into mature miRNAs. The processing of pri-miRNAs is the most highly regulated step in the miRNA biogenesis pathway. Therefore, understanding the molecular mechanisms of pri-miRNA processing and its regulation represents a very important objective in the miRNA filed. Recent studies suggest that the Drosha-DGCR8 complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here, we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the Microprocessor and promotes global miRNA biogenesis in HeLa cells. Accordingly, chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 & Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 exhibits a novel pri-miRNA binding activity and promotes the Drosha-pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by resulting in premature release of pri-miRNA transcripts from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work expands the functional repertoire of the H1 family of proteins and suggests a new concept of chromatin retention factor for widespread co-transcriptional miRNA processing.Item Identification of Smaller Noncoding RNAs Produced by Mycobacterium Tuberculosis in Infected Macrophages That Regulate Mtb Growth and Survival(2021-12-10) Coskun, Fatma Sevde; Hancks, Dustin C.; Conrad, Nicholas; Winter, Sebastian E.; van Oers, Nicolai S. C.It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different proteins encoded by Mtb in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. A subset includes small RNAs (sRNAs) between the sizes of 50-350 nts. The current study focused on the identification and characterization of miRNA-like sRNAs <50 nts produced by Mtb. A sRNA-centered RNA-sequencing approach was performed and a subset of Mtb-encoded smaller noncoding RNAs (sncRNAs) were identified. Thirty-five distinct Mtb-encoded sncRNAs were discovered, with most being induced in infected eukaryotic cells. Three sncRNAs, sncRNA-1, sncRNA-6, and sncRNA-8, predominated the read counts. They were contained in longer RNA transcripts with stable secondary RNA stem loops and structures like precursor microRNAs. My work established that sncRNA-1 positively regulates two mycobacterial transcripts involved in oleic acid biosynthesis. Loss- and gain- of-function approaches reveal that sncRNA-1 enhances Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Given evidence that RNA processing enzymes were involved in the formation of the sncRNAs, different components of core RNA degradosome were characterized for their ability to process the precursor forms of the sncRNAs. My work revealed that PNPase degrades sncRNA-8 and preliminary evidence suggests that sncRNA-1 is also likely a target, which could be critical in the oleic acid deficient media. Overall, my study reveals that Mtb produces a set of sncRNAs in infected cells, with one modulating mycobacterial gene expression and mycobacterial pathogenicity coupled to oleic acid biogenesis. Future studies will address the functions of other sncRNAs and focus on the identification of sncRNA processing enzymes.Item Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer(2013-07-24) Borkowski, Robert John; Scaglioni, Pier Paolo; Pertsemlidis, Alexander; White, Michael A.; Cobb, Melanie H.; Corey, David R.Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related fatalities in the US. This is due in part to a lack of highly effective therapies for advanced cases, and this is of special concern as most NSCLC cases are not diagnosed until they are in an advanced, later stage. Recent successes in developing genotypically-targeted therapies with potency only in a well-defined subpopulation of tumors suggests that identifying targeted therapies for additional common NSCLC genotypes will improve patient survival. In this study I utilized a library of inhibitors to microRNAs, a class of post-transcriptional gene regulators, to identify novel synthetic lethal miRNA inhibition:molecular mechanism interactions in NSCLC. I accomplished this by screening a panel of 13 NSCLC and immortalized normal lung epithelium (HBEC) cell lines in two phases to identify miRNA inhibitors with selective toxicity in the NSCLC cell lines that were also benign in an HBEC cell line. Two inhibitors, the miR-92a and miR-1226* inhibitors, met these criteria. I then collected toxicity data in an expanded panel of 29 total cell lines. This expanded toxicity data was used to identify p53 loss as a molecular mechanism correlated with sensitivity to the miR-92a and miR-1226* inhibitors in NSCLC cell lines. This was recapitulated by demonstrating sensitivity after knockdown of p53 in the previously resistant HBEC30KT cell line. I determined that the inhibitors were toxic in a very sequence-specific manner and that they down-regulated the miR-17~92 polycistron. Down-regulation of the polycistron was toxic in a context-specific manner, and the down-regulation of the miR-17~92 cluster in sensitive cell lines mimicked activation of a 1α, 25-dihydroxyvitamin D3 response in NSCLC cell lines in a manner consistent with sensitivity to the miR-92a inhibitor. The results of this investigation demonstrate that the screening approach utilized in this study was capable of identifying a synthetic lethal miRNA inhibition:molecular mechanism interaction, and that I was then able to use a genetically defined model of the mechanism to identify a relevant mechanism of action for the toxic inhibitors.Item Identifying Novel Regulators of LIN28B Through a Genome-Wide CRISPR/Cas9 Screen(2017-01-17) Budhipramono, Albert; Hao, Zhu; Nguyen, LiemLIN28 is a family of RNA-binding proteins that are well-conserved across species. It is well-known to regulate developmental timing by inhibiting the biogenesis of the let-7 microRNAs. Numerous studies have shown that LIN28 is dysregulated in a wide spectrum of cancer types, especially pediatric cancers such as hepatoblastoma and Wilms' tumor. Our laboratory previously showed that reactivation of LIN28B, one of the two LIN28 homologs, is sufficient to drive liver cancer, and that LIN28B deletion is detrimental to tumor development. LIN28B exerts its oncogenic function by inhibiting the maturation of let-7 precursors, as well as directly binding to and enhancing translation of growth-promoting mRNA targets, such as members of the IGF2BP family. While our work and others established that LIN28B functions as an oncogene, the identity of factors that regulate LIN28B expression during normal development and cancer remains elusive. As LIN28B is a driver of oncogenesis in various cancers, understanding its regulation in the process of oncogenesis will help uncover novel therapeutic targets. Here, we show an original approach for identifying regulators of the human LIN28B gene utilizing the CRISPR/Cas9 genome engineering system. Traditional transgenic approaches to study gene function often fail to capture transcriptional regulation at distal promoter and enhancer sequences. Using the CRISPR/Cas9 system, we knocked a GFP reporter sequence into the endogenous locus of LIN28B in human cancer cell lines, engineering a fusion LIN28B-GFP protein. This approach is unique in that GFP expression will be altered not only by changes in regulation at the coding sequence, mRNA and protein levels, but also changes at distal regulatory sequences. To identify unknown regulators of LIN28B, we will perform a genome-wide CRISPR/Cas9-mediated knockout screen in human cells expressing the fusion LIN28B-GFP protein. Using a genome-scale library with 76,441 sgRNAs, we will knock out 19,114 genes individually and assess their effects on LIN28B levels by measuring GFP expression. sgRNAs that are enriched in the high GFP-expressing population suggest genes that normally function as inhibitors of LIN28B. On the other hand, sgRNAs that are depleted suggest activators of LIN28B. Through this screen, we hope to gain further insight into how LIN28B is regulated in normal development and cancer. Furthermore, identifying regulators of LIN28B can provide novel avenues for developing cancer therapeutics.Item Mapping the Landscape of Acquired Vulnerabilities in Ovarian Cancer(2013-06-05) Shields, Benjamin Baker; Castrillon, Diego H.; White, Michael A.; Altschuler, Steven J.; Brekken, Rolf A.Recent undertakings to identify the genetic lesions associated with ovarian cancer have noted the striking diversity of mutations occurring in this disease. This genetic diversity has complicated the search for novel therapies. However, recent data has suggested that one commonality of ovarian tumors might be ablation of miRNA biogenesis. Here I conducted a broad-scale gain-of-function microRNA (miRNA) screen in 16 ovarian cancer cell lines to annotate the functional landscape present in such a chaotic genetic background. miRNAs function as multigenic perturbations allowing for interrogation of maximal gene space with few experiments. This screen identified multiple miRNAs reducing cell viability with the majority of hits being toxic in only one or two lines screened. This surprising finding reflected the commonality of altered miRNA function in ovarian tumors while also suggesting that specifics of this alteration in function are unique to each tumor. To investigate more public vulnerabilities, I focused mechanistic studies on miRNAs displaying penetrance in greater than 5 cell lines. miR-517a reduced cell viability in over 30% of the panel and also reduced tumor burden in vivo. Functional analysis of the predicted targets of miR-517a revealed that expression of this miRNA reduced protein levels of ARCN1, a member of the coatamer complex, and that knockdown of ARCN1 reduced cell viability similar to miR-517a. Another penetrant miRNA, miR-124a, reduced cell viability in 37.5% of the panel and functional analysis of this miRNA revealed it promoted a cell differentiation program. Analysis of predicted targets revealed that expression of miR-124a reduced expression the homeodomain transcription factor SIX4, resulting in increased signaling along the tumor suppressive AMPK pathway and epithelial differentiation. Furthermore, SIX4 displayed increased expression in ovarian tumors and depletion of SIX4 expression reduced tumor cell viability in vitro and in vivo. Therefore, SIX4 overexpression might function to deflect cell differentiation in tumors. Thus, the common loss of miRNA function observed in ovarian tumors might serve to maintain an undifferentiated state, and engagement of cell fate determination programs via re-expression of miRNAs can result in catastrophic consequences for cancer cell viability.Item Mechanisms of p19Arf-Mediated Regulation of Perivascular Cell Biology During Mammalian Eye Development(2015-08-03) Iqbal, Nida S.; Johnson, Jane E.; Mendell, Joshua T.; Gupta, Rana K.Since its discovery in 1995, p19Arf has been under critical interrogation for its role as a potent cell cycle regulator and tumor suppressor. In the last decade, there has been considerable evidence describing an essential function for p19Arf during mammalian eye development. In this context, p19Arf is required for the ultimate involution of the hyaloid vasculature system that exists in the primary vitreous space and serves to nourish the lens and retina. Knock-out mouse models for p19Arf demonstrate that in the absence of Arf, there is an abnormal accumulation of cells that persist into the adult secondary vitreous and cause detrimental ocular defects including blindness, retinal detachment and lens opacity. It has been further demonstrated that p19Arf enacts a dual mechanism to inhibit the accumulation of the perivascular cells that occupy the vitreous space during development and lead to the clearing of these cells followed by eventual involution of the underlying vasculature system. Platelet-derived growth factor receptor β (Pdgfrβ) is required for the accumulation of cells in the absence of Arf and while it is clear that p19Arf utilizes a p53-dependent mechanism to inhibit Pdgfrβ transcription, the mechanism by which it can inhibit Pdgfrβ protein in the absence of p53 is not well defined. Further, the biological consequences of Arf expression have, to date, only been studied in a context in which Arf is not normally expressed, such as during tumor progression and culture shock. This work addresses these two open questions. First, I will discuss a novel capacity for p19Arf to employ microRNAs outside of the p53 pathway to lead to repression of Pdgfrβ protein. Next I will describe an ex vivo cell culture system to study unexplored facets of Arf biology in a context in which it is endogenously expressed.Item MicroRNAs and Related Tissue Remodeling Genes in Rotator Cuff Repair in a Rat Model(2015-01-26) Contreras, Erik; Wei, Fuxin; Shirley, Zachary; Shelton, William; Khazzam, Michael; Chen, Christopher T.The majority of rotator cuff tears seen clinically are atraumatic and chronic in nature, which can contribute to joint inflammation and tendon degeneration. Although some inflammation is vital to the healing process, excessive inflammation and high levels of matrix-degrading enzymes have been associated with impaired healing of the rotator cuff. The underlying factors regulating the level of inflammation in the injured rotator cuff still remain unclear. The purpose of this study was to investigate the expression of miRNAs in the setting of acute and delayed repair of a torn rotator cuff in a rat model. We hypothesized that the expression of miRNAs related to the inflammatory process would be altered in the injured rotator cuff compared to the control. Fourteen rats were randomized to either acute or delayed rotator cuff repair. Seven animals underwent detachment of the supraspinatus and infraspinatus tendons followed by immediate repair, while the remaining seven underwent delayed repair 2 weeks later. Sham surgery on the contralateral shoulder was used as a control. mRNA expression for COL1A1, COL3A1, TGF-β1, MMP-9, MMP-13 and IL-6 was determined and select samples were analyzed for miRNA profiles. Upon analysis, 57 miRNAs showed significant differences between groups, including 14 miRNAs with significant differences between acute and delayed repair groups (p<0.05). miRNA correlation analysis with mRNAs expression revealed two distinct groupings. The first grouping includes IL-6, MMP-13, and MMP-9, genes that are traditionally associated with inflammation and matrix degradation. The second grouping contains TGFβ1 and COL1A1, genes that are traditionally associated with the repair process. These findings suggest that groups of miRNA expression may play a more global role in regulating the balance between the inflammatory and repair processes, although any specific interactions have yet to be determined. Interestingly, IL-6 has been shown to promote increased levels of miR-18a* expression in hepatocytes, where it then acts in a positive feedback loop of the IL-6 signaling pathway. Our data, which shows a positive correlation between IL-6 and miR-18a* (R2=0.63), is consistent with these findings. We speculate that there may be a similar interaction between IL-6, miR-18a*, and the expression of downstream targets involved in rotator cuff inflammation and repair. These findings support our hypothesis that the expression of miRNAs related to the inflammatory process is significantly altered in the injured rotator cuff compared to the control tendon. Of particular interest is miR-18a*, which has been shown to increase levels of matrix-degrading enzymes, pro-inflammatory cytokines, and chemokines in arthritis synovial fibroblasts. This will require further experimentation in order to determine the precise gene targets and whether or not down-regulation of miR-18a* could lead to a mechanism by which one could decreaseItem MicroRNAs: new drug targets for kidney diseases(2018-01-26) Patel, VishalItem MicroRNAs: Tissue Expression and Role in 3T3-L1 Pre-Adipocyte Differentiation(2009-01-14) Sun, Tingwan; Mangelsdorf, David J.MicroRNAs, which are endogenous small RNAs around 22 nucleotides, play important roles in many physiological processes. We investigated whether microRNAs regulate 3T3-L1 pre-adipocyte differentiation. The expression of microRNAs during 3T3-L1 pre-adipocyte differentiation was detected by microarrays and confirmed by northern blot and quantitative real time PCR. Several microRNAs, including let-7, were up-regulated at the late stage of 3T3-L1 adipogenesis. let-7 expression specifically increased during the late stage of 3T3-L1 differentiation and ectopic introduction of let-7 in 3T3-L1 cells before hormonal induction inhibited 3T3-L1 adipogenesis. Both the mRNA and protein levels of HMGA2, a target of let-7, decreased with ectopic let-7 presence in 3T3-L1 cells. Also, HMGA2 protein level was inversely correlated to let-7 levels during 3T3-L1 adipogenesis. Knock-down of Hmga2 or E2f1 by siRNA inhibited 3T3-L1 pre-adipocyte differentiation. Our results suggest let-7 can stop clonal expansion of 3T3-L1 cells and bring them to final growth arrest and terminal differentiation by targeting Hmga2. In an effort to explore the role of microRNA, the expression of 111 microRNAs in 36 mouse tissues was detected by quantitative real time PCR. MicroRNAs either show universal expression in all tissues or specific expression in certain tissues, suggesting their roles in these tissues. Different isotypes of the same microRNAs or microRNAs transcribed from the same genomic location show similar expression pattern in mouse tissues. Hierarchical cluster analysis based on the expression of microRNA in tissues showed that tissues having similar physiologic functions or anatomic locations clustered together, suggesting the roles of microRNAs might be consistent with the functions of the tissues in which they are expressed. Comparison of the expression of microRNAs with that of nuclear receptors in mouse tissues showed positive correlations between a select number of nuclear receptors and microRNAs, but these relationships need to be verified by experimental data. In all, the expression profile of microRNAs in mouse tissues provides a useful tool for microRNA studies.Item MiR-10a Regulation of Drug Response and Cancer Stem Cell Populations in Non-Small Cell Lung Carcinomas(2013-09-26) DeSevo, Christopher Gerard; Castrillon, Diego H.; Shay, Jerry W.; Scaglioni, Pier Paolo; Pertsemlidis, Alexander; Minna, John D.Phosphatidylinositol 3-kinases (PI3Ks) are enzymes involved in diverse cellular functions including cell growth, proliferation, differentiation, motility, survival and apoptosis. Many of these functions relate to class I PI3Ks, heterodimers composed of regulatory and catalytic subunits that convert extracellular cues to intracellular responses upon activation. Overall, this signaling pathway is under tight regulation and even slight perturbations can lead to aberrant pathway activation. In NSCLC cell lines, we found that manipulation of miR-10a results in significant changes to both mRNA and protein levels of PI3K. In the context of cellular response to front-line chemotherapeutic agents used to treat NSCLC, I uncovered that miR-10a mimic decreases cell viability10-fold in the presence of paclitaxel relative to drug alone, while inhibiting miR-10a results in a 10-fold increase, suggesting that high levels of miR-10a may be predictive of response to such agents. To assess its prognostic value, we interrogated miR-10a expression in NSCLC tumors and found that high miR-10a levels correlate with longer overall patient survival. miRNAs can target hundreds of genes, meaning that miR-10a may regulate PIK3CA expression both directly and indirectly. We identified the transcription factor GATA6 as both a target of miR-10a with a predicted miR-10a target site in its 3’UTR and a regulator of PI3K expression, with several conserved binding sites in the promoter of PIK3CA. These findings demonstrate that miR-10a regulates the PI3K pathway at two distinct levels. Microarray expression profiling of NSCLC cells treated with miR-10a mimic had significant down regulation of ALDH1A3, a marker of cancer stem cells. This relationship was confirmed through functional validation of ALDH activity. Multiple miRNA target prediction algorithms showed that ALDH1A3 is not a direct target of miR-10a. To uncover the direct target of miR-10a we used a targeted siRNA screen containing genes implicated in stem cell maintenance to reveal that the WNT and Notch pathway are important for cell survival. Both pathways are down-regulated when cells are treated with a miR-10a mimic. Bioinformatic analysis identified DVL3 as a miR-10a target gene. Manipulation of miR-10a levels resulted in significant changes in both mRNA and protein levels of DVL3. Finally, loss of DVL3 expression significantly decreased ALDH1A3 protein levels and the population of ALDH+ cells. Collectively, my work has uncovered miR-10a as mediator of the potent PI3K oncogenic pathway through both direct and indirect mechanisms, a modulator of cellular response to paclitaxel and finally its identification in NSCLC stem cell maintenance through regulation of the WNT and NOTCH pathways.Item Post-Transcriptional Regulation by microRNAS in Pregnancy and Parturition(2012-08-15) Renthal, Nora Edwards; Mendelson, Carole R.Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, while spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and negatively impact PR function. In this study, we uncovered a new regulatory pathway whereby miRNAs serve as hormonally-modulated and conserved mediators of contractile gene regulation in the pregnant uterus from mouse to human. Using miRNA and gene expression microarray analyses of uterine tissues, we identified a conserved family of miRNAs, the miR-200 family, that is highly induced at term in both mice and humans, as well as two coordinately downregulated targets, zinc finger E-box binding homeobox proteins, ZEB1 and ZEB2, which act as transcriptional repressors. We also observed upregulation of the miR-200 family and downregulation of ZEB1 and ZEB2 in two different mouse models of preterm labor. We further demonstrated that ZEB1 is directly upregulated by the action of P4/PR at the ZEB1 promoter. Excitingly, we observed that ZEB1 and ZEB2 inhibited expression of the contraction-associated genes, oxytocin receptor and connexin-43 and blocked oxytocin-induced contractility in human myometrial cells. Together, these findings implicate the miR-200 family and their targets ZEB1 and ZEB2 as novel P4/PR-mediated regulators of uterine quiescence and contractility during pregnancy and labor, and shed new light on the molecular mechanisms involved in preterm birth.Item Regulation of Cellular Growth and Differentiation by MicroRNAs -21 and -451(2013-05-31) Patrick, David M.; Olson, Eric N.; MacDonald, Raymond J.; Hill, Joseph A.; Johnson, Jane E.; Huang, LilyMicroRNAs are small RNAs approximately 20-24 nucleotides in length that are conserved throughout evolution. MicroRNA genes are transcribed by RNA polymerase II and are processed both in the nucleus and the cytoplasm from longer precursor RNAs. Functionally, microRNAs interact with Argonaute proteins and guide the formation of a complex with messenger RNAs by Watson-Crick base-pair formation between the microRNA and mRNA. This association stimulates the formation of the microRNA-RNA-induced silencing complex which, upon association with essential adaptor molecules such as GW182, recruits transcriptional repressors and mRNA destabilizers. Essential developmental processes such as embryonic stem cell differentiation and cardiovascular development have been shown to be dependent upon microRNAs. MicroRNAs also participate in a variety of disease processes including tumorigenesis and cardiovascular disease. MicroRNA-451 (miR-451) is regulated during erythrocyte terminal differentiation. The expression of miR-451 is restricted to late erythrocyte precursors and terminally differentiated erythrocytes. We therefore hypothesized that miR-451 plays a role in terminal erythroid differentiation. Deletion of miR-451 in mice results in a terminal erythroid differentiation defect both embryonically and in adulthood. These animals display a reduction in hematocrit and an inability to sustain a high erythropoietic rate. Transient inhibition of miR-451 results in the same defect. Transcript profiling of miR-451-/- erythroblasts revealed upregulation of 14-3-3ξ, a molecule implicated in the regulation of hematopoiesis. Knockdown of 14-3-3ξ with shRNA in miR-451-/- erythroblasts attenuates the differentiation defect. These data show the essential role of miR-451 repression of 14-3-3ξ during terminal erythrocyte differentiation. Finally, the potent effect of miR-451 inhibition on erythrocyte production suggests that this strategy may be efficacious for the treatment of polycythemia vera, a myeloproliferative neoplasm characterzed by excessive erythrocyte production. Inhibition of miR-451 in a mouse model of PV significantly reduces disease burden. MicroRNA-21 (miR-21) is regulated in a variety of both human and mouse models of disease. MiR-21 has been widely reported as a driver of tumorigenesis and is consistently upregulated in cardiac remodeling. It has been suggested that miR-21 plays a protective role during cardiac hypertrophy, however, an opposing report suggests that miR-21 inhibition is beneficial in a mouse model of cardiac remodeling. We therefore hypothesized that miR-21 played an essential role in cardiac hypertrophy and remodeling. Deletion of miR-21 in mice resulted in no observable phenotype. MiR-21-/- displayed cardiac remodeling, cardiac stress-responsive gene activation, and reduction in cardiac function in response to four cardiac stress models: thoracic aortic constriction, angiotensin II infusion, calcineurin overexpression, and myocardial infarction. Moreover, inhibition of miR-21 with an LNA-modified miR-21 inhibitor did not modify cardiac remodeling. Finally, inducible genetic deletion of miR-21 did not modify the cardiac response to TAC. These data do not support a role for miR-21 in cardiac disease, however, further analyses of miR-21-/- mice show that these animals are protected from non-small-cell lung cancer (NSCLC). Furthermore, miR-21-/- mouse embryonic fibroblasts are sensitized to doxorubicin-induced apoptosis. These data suggest that inhibition of miR-21 will be efficacious in the treatment of NSCLC while having minimal effects on other tissue types.Item Regulation of Exercise-Dependent Cardiac Growth by MicroRNAs(2013-01-16) Johnson, Brett A.; Olson, Eric N.The heart is an adaptive organ which undergoes pathological or physiological remodeling in response to a variety of stimuli to meet the demands of the body. Chronic exercise training promotes a physiological remodeling response in which the heart increases in size to match loading demands. In this thesis, I present my studies on the function of microRNAs during exercise-induced cardiac remodeling. First, I show the expression of muscle-specific microRNA (miRNA), miR-499, is down-regulated by voluntary free-wheel running in hearts of mice. I hypothesized the reduction of miR-499 may be required for exercise-induced cardiac hypertrophy. I found forced cardiac over-expression of miR-499 was associated with diminished physiological cardiac growth, whereas genetic deletion and antimiR mediated inhibition of miR-499 caused enhanced physiological growth following exercise. I also explored the mechanism by which miR-499 represses exercise-induced cardiac growth. I determined the repressive effects of miR-499 are mediated through regulation of IGF-1/PI-3K/Akt and beta-catenin signaling pathways, which drive physiological growth of the heart. I demonstrated the effects of miR-499 on physiological cardiac growth are mediated, at least in part, through repression of a network of genes including p85-alpha, Rictor, Lin7c and Fzd4. Collectively, the results of my thesis research identify miR-499 as a pivotal regulator of exercise-induced cardiac hypertrophy.Item Regulation of Metabolic Processes by Micrornas and Class I Histone Deacetylases(2013-01-17) Carrer, Michele; Olson, Eric N.Obesity is a medical condition resulting from accumulation of excess body fat that affects more than 30% of the adult population in the U.S. Obesity-related pathological conditions include heart disease, stroke, type 2 diabetes and certain types of cancer. Despite the high incidence and the elevated social costs, the molecular basis of obesity and associated metabolic syndrome are still poorly understood. Yet, the need for novel therapeutic approaches for the treatment and prevention of obesity remains. In humans and animal model of disease, hallmarks of obesity include dysregulation of genes involved in mitochondrial function, lipid uptake and lipid storage. The dynamic and modifiable regulation of transcriptional pathways that control mitochondrial function and adipogenesis, as well as additional aspects of mammalian metabolism, will provide new approaches for pharmacological intervention in obesity. Thus, the modulation of epigenetic histone modifications and microRNA functions represents a potentially powerful approach for the treatment of metabolic disorders. We show that the Ppargc1b gene, which encodes the PGC-1β protein, also co-transcribes two microRNAs, miR-378 and miR-378*. Mice lacking miR-378/378* are resistant to high fat diet-induced obesity and display enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. Taken together, our findings reveal that miR-378 and miR-378* function as integral components of the regulatory circuit formed by PGC-1beta and nuclear hormone receptors to control the overall oxidative capacity and energy homeostasis of insulin-target tissues. MiR-378/378* mutant mice do not display overt phenotypes under normal laboratory conditions, whereas their phenotypes become apparent under conditions of stress, in this case in response to excessive caloric intake. Thus, pharmacological modulation of miR-378/378* function might represent an effective approach in the treatment of obesity. In obese humans and mice, the unused caloric energy resulting from excessive net caloric intake is converted to triglycerides and stored in adipocytes for further usage. Lipid accumulation within adipocytes is under the control of a cascade of transcription factors that interact with histone acetyltransferases and deacetylases. We show that histone deacetylase inhibitors efficiently block adipocyte differentiation in vitro. Furthermore, through a loss-of-function approach, we provide evidence that histone deacetylases 1 and 2 play redundant and requisite roles in adipogenesis. In conclusion, we unveiled previously unrecognized roles for miR-378/378* in the control of mitochondrial metabolism and energy homeostasis, and for histone deacetylases in the control of adipocyte differentiation.Item Regulation of Skeletal Muscle Innervation and ALS Pathogenesis by MicroRNA 206(2010-01-12) Williams, Andrew H.; Olson, Eric N.Motor neurons and the skeletal muscle fibers they innervate maintain an intimate relationship that requires bidirectional signaling for the establishment and maintenance of neuromuscular synapses and muscle function. Abnormalities in the regulation of neuromuscular gene expression often result in neuropathies and myopathies, reflecting the intimate communication between muscle and motor nerve. In this thesis, I present my studies on the function of microRNAs in neuromuscular synapse regeneration and neurodegenerative disease. First, I show that the expression of a muscle-specific microRNA (miRNA), miR-206, is dramatically upregulated following surgical denervation of skeletal muscle and in a mouse model of amyotrophic lateral sclerosis (ALS). The responsiveness of the miR-206 gene to the state of motor innervation is dependent on binding sites for MyoD in an upstream enhancer. Based on the upregulation of miR-206 following denervation and its synapse-enriched expression pattern, I hypothesized that miR-206 is an important regulator of neuromuscular junction (NMJ) physiology and I generated miR-206 mutant mice. Using these mice, I demonstrated that miR-206 is an essential regulator of neuromuscular synapse reinnervation following nerve injury. The requirement of miR-206 for efficient reinnervation reflects, at least in part, its repressive influence on histone deacetylase 4 (HDAC4). I also explored another function of miR-206, as an essential modulator of retrograde growth factor signaling during the progression of neurodegenerative disease. By crossing miR-206 mutant mice to G93A-SOD1 transgenic mice, which express a mutant form of superoxide dismutase (SOD), I determined that the loss of miR-206 accelerates the pathogenesis of ALS due to the loss of functional NMJs. Thus, the results of my thesis research demonstrate that miR-206 functions as a sensor of motor innervation and regulates a retrograde signaling pathway required for nerve-muscle interactions during stress and disease.