Browsing by Subject "microRNAs"
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Item Determine the spatial and temporal expression patterns of Drosophila miRNAs(2007-12) Ao, Xue, 1977-; Macdonald, Paul M.microRNAs (miRNAs), are a class of small (21-23nt), endogenous, non-coding RNAs which regulate gene expression. In animals, most miRNAs recognize their mRNA targets though imperfect base paring with complementary sites at the 3’UTR of the mRNAs, resulting in translational repression of the target genes. The specific functions of miRNAs in Drosophila are generally unknown. To determine the spatial and temporal expression patterns of miRNAs, it is necessary to identify both the sites and time of miRNA action and further elucidate miRNA functions. I made a series of GFP reporter transgenes (miRNA sensors) in UAS vector containing multiple copies of synthesized miRNA targets in the 3’UTR. The sensors were expressed in a range of tissues driven by different GAL4 drivers. We expected that our sensors would reveal when and where miRNAs are actively regulate gene expression. However, the sensor strategy failed to detect clear expression patterns of the miRNAs we tried.Item microRNA-223 Regulates Macrophage Polarization and Diet-induced Insulin Resistance(2013-05-01) Meng, CongMacrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulinresistance. miR-223-/- mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell-mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow-derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic pro-inflammatory pathways and enhances the alternative anti-inflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. For the first time, this study demonstrates that miR-223 acts to inhibit Pknox 1,suppressing pro-inflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance.Item Modulation of Intestinal Micrornas by a Chemoprotective Diet(2012-12-05) Shah, Manasvi Shailesh 1984-We have hypothesized that dietary modulation of intestinal miRNA expression may contribute to the chemoprotective effects of nutritional bioactives (fish oil and pectin). Using a rat colon carcinogen model, we determined miRNAs-let-7d, miR-15b, miR-107, miR-191 and miR-324-5p were modulated by fish oil + pectin. We also demonstrated that BACE1 and PTEN are targets of miR-107 and miR-21, respectively. To further elucidate the biological effects of diet and carcinogen on miRNAs, we integrated global miRNAs, total and polysomal gene expression datasets obtained from the above mentioned study and used four computational approaches. We demonstrated that polysomal profiling is tightly related to microRNA changes when compared with total mRNA profiling. In addition, diet and carcinogen exposure modulated a number of microRNAs and complementary gene expression analyses showed that oncogenic PTK2B, PDE4B, and TCF4 were suppressed by the chemoprotective diet at both the mRNA and protein levels. To determine the function of select diet and colon carcinogen modulated miRNAs and to validate their targets, we carried out a series of loss and gain of function experiments along with luciferase reporter assays. We verified that PDE4B and TCF4 are direct targets of miR-26b and miR-203, respectively. PTK2B was determined to be an indirect target of miR-19b. In addition, microRNA physiological function was assessed by examining effects on apoptosis and cell proliferation. To better understand how the colonic stem cell population responds to environmental factors such as diet and carcinogen, we investigated the chemoprotective effects of dietary agents on miRNAs in colonic stem cells obtained from Lgr5-EGFP-IRES-creERT2 knock in mice injected with AOM. We demonstrated that based on relative expression of miR-125a-5p, miR-190b and miR-191 in stem cells vs. daughter cells and differentiated cells, these miRNAs may be stem cell specific miRNAs. We also identified miR-21 to be significantly reduced in stem cells compared to differentiated cells and selectively modulated by these dietary agents in stem cells. In summary, our results indicate for the first time that fish oil plus pectin protect against colon tumorigenesis in part by modulating a subset of miRNAs and their target genes (mRNAs) implicated in the regulation of the colon stem cell niche and tumor development.