Browsing by Subject "microRNA"
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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 Effects of Exercise and Diet- induced Weight Loss in Overweight/Obese Women on Characterization of Serum/White Blood Cells, microRNAs and Cytokine Gene Transcription(2013-12-11) Simbo, Sunday YamoThis study examined the effects of exercise and diet-induced weight loss on markers of inflammation in obese females. Forty-nine obese women (age 48.2?10.5 years, height 63.5?2.7cm; weight 203.3?30.5 kg; BMI 35.6?5.6 kg/m^(2); 45.9?4.4 % body fat) completed a 12-week study (exercise group (EX): n=29; control group (C): n=20). Participants followed an energy-restricted diet (1,200 kcal/d for 1-week and 1,500 kcal/d for 11 weeks; 30% CHO, 45% P, and 25% F) while participating in a 30-min circuit resistance-training (3 days/wk) and 30 min walking program on non-workout days. DEXA body composition, fitness, and serum/white blood cells samples were obtained at 0, 4, 8 and 12 wks. The expression of microRNA (21 and 146a) and the expression levels of IL-6, TNF-?, (PTEN, TRAF6)/PI3k/AKT/NF-kB were measured by real-time RT-PCR at 0 and 12 wks. Data were analyzed by MANOVA and presented as changes from baseline after 12 wks for the EX and C groups. Overall MANOVA analysis revealed a significant time effect (p=0.004) and group x time effect (p=0.004) for body composition measures. Participants in the EX group experienced significant changes in body weight (EX -4.0?4.4 kg; C 0.1?3.0 kg, p=0.001), fat mass (EX -3.8?4.0 kg; C -0.03?2.0 kg, p<0.001), and percent body fat (EX -2.7?3.4%; C -0.1?1.7%, p=0.002). Overall MANOVA analysis revealed a significant time effect (p<0.001) and group x time effect (p=0.003) for measures of fitness. Overall significant MANOVA interaction was observed among EX and C groups (Wilks? Lambda p<0.001) on markers of inflammation. Significant interactions were observed among groups in microRNA 21 (EX -1.5?2.34; C 0.13?2.2, p=0.03), mRNA expression levels of PTEN (EX -4.5?3.2; C -1.6?3.4, p=0.005), IL-6 (EX -2.8?3.6; C 2.8?2.2, p=0.00); and, TNF-? (EX -0.52?2.5; C 2.3?1.9, p=0.00). Changes in microRNA (21 and 146a) were positively and significantly correlated to body weight, total weight, fat mass, and body fat percent, with circulatory levels of IL-6 and TNF-?. Results indicate that 12-wks of participation in an exercise and weight loss program significantly affects microRNA 21 and its target gene PTEN, mRNA TNF-?, and mRNA IL-6 levels suggesting an anti-inflammatory response compared to a control group.Item Phytochemistry and Health Benefits of Grapes and Wines Relevant to the State of Texas(2012-10-19) Del Follo Martinez, ArmandoThe overall objective of this work was to increase the knowledge regarding American hybrid grapes and wine-making techniques relevant to the State of Texas, specifically to investigate grape chemistry of hybrid grapes, to evaluate the effects of micro-oxygenation on wine chemistry, and to elucidate anti-cancer effects of wine compounds and extract in colon cancer cells in vitro. The methods used include HPLC-PDA-EIS-MSn and molecular bioassays. The American hybrid grapes, Black Spanish (Vitis aestivalis hybrid) and Blanc Du Bois (Vitis aestivalis hybrid), were compared to Cabernet Sauvignon and Merlot (Vitis vinifera) in their phytochemical composition. Total phenolics were similar in red grape varieties, but lower in white grapes. In Black Spanish grapes, anthocyanins and antioxidant capacity (ORAC) exhibited the highest values. Non-anthocyanin polyphenolics did not show qualitative differences in the four grape varieties. The presence of anthocyanins diglucosides was unique to Black Spanish grapes. The second experiment involved application of micro-oxygenation with oak inner staves to evaluate the effect of this new vinification technology on the stability of anthocyanins. Overall, anthocyanins exhibited significant decreases over time in the following order: control, wine with oak pieces, oak barrel, and micro-oxygenation. The anti-cancer effect of a combination of wine compounds, resveratrol/quercetin (RQ), and a polyphenolic extract from Black Spanish wine were investigated in colon cancer cells HT-29. RQ reduced the generation of reactive oxygen species (ROS), whereas the ORAC increased. RQ reduced cancer cell viability and proliferation, induced caspase-3-cleavage, and increased PARP-cleavage. Additionally, Sp1, Sp3, Sp4, and survivin were down-regulated at mRNA and protein levels. Furthermore, RQ decreased microRNA-27a (miR-27a) and induced ZBTB10, suggesting that RQ interactions with the miR-27a-ZBTB10-axis play a role in Sp down-regulation. Similar results were obtained for the wine extract. This work will provide valuable information regarding grape varieties, potential health benefits of wine, and wine production techniques to the wine industry in Texas and beyond.Item The Effect of Disrupted Circadian Rhythm and Associated microRNA on Biliary Injury and Malignant Transformation(2014-12-12) Han, YuyanCholangiocarcinoma (CCA) is a devastating tumor characterized by late presentation of symptoms with limited treatment options. Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that trigger mRNA translation, repression or degradation. The aim of the study was to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Human intra- and extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hours before stimulation with 50% serum for 2 hours. The 24-hours rhythmic expression of core clock genes, such as Per1/2/3, CLOCK, Bmal1, Cry1/2 and two clock-controlled genes (CCGs) WEE1 and DBP, was evaluated in the selected CCA cells and H69 controls by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting Mz-ChA-1 CCA cells with Per1 or empty vector. In parallel studies, we silenced miR-34a expression with anti-miR-34a inhibitor. Then, we measured: (i) cell proliferation by MTS assays and PCNA immunoblots; (ii) cell cycle; (iii) apoptosis; and (iv) cell migration and. We used luciferase assay to demonstrate whether Per1 acts as a direct target of miR-34a. Finally, we maintained CCA xenograft nude mice in complete dark or light/dark cycle for up to 40 days before evaluating tumor growth. We found the 24-hours rhythmical expression of Per1 was abolished in all CCA cell lines. The rhythmic expression of Bmal1, CLOCK, Per2/3, Cry1/2, WEE1 and DBP was also lost in some of the CCA cell lines tested. After overexpression of Per1, Mz-ChA-1 showed: (i) reduced cell proliferation; (ii) higher G0/G1 arrest and lower G2/M arrest and (iii) enhanced apoptosis. miR-34a was rhythmically expressed in CCA cell lines and H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in the selected CCA cell lines. Per1 was verified as a target of miR-34a. However, prolonged darkness therapy did not inhibit the CCA xenograft growth in vivo. Summary and conclusions: Disruption of circadian rhythms contributes to the malignant phenotypes of human CCA, and may serve as novel prognostic or therapeutic targets for CCA.