Browsing by Subject "Per1"
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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.Item Timing Matters: The Role of Circadian Clock Genes In Development and Toxin Responses(2009-05-15) Qu, XiaoyuMost members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.