Project description:In this study, breast cancer MCF-7 cells were cultured under 0.5% oxygen for 24 h followed by 24 h of reoxygenation. Cells was harvested at 0, 1, 12, and 24 h during reoxygenation, and examined the miRNA profile by Nanostring nCounter. Forty-three miRNAs had dramatic changes as compared with 0 h upon reoxygenation, with 63% (n=27) of the miRNAs up-regulated upon reoxygenation. Among these miRNAs, miR-769-3p, which was down-regulated in MCF-7 upon reoxygenation, was chosen for further investigation. The hypothesis was that the down-regulation of NDRG1 upon reoxygenation was regulated by miRNAs. To examine wether miRNAs regulate NDRG1 under 0.5% O2 concentrations, the miRNA expression profiles of MCF-7 cells under reoxygenation were examined. Cells were harvested at 0 (hypoxia control), 1, 12, and 24 h upon reoxygenation. Each profile was done in triplicate. The genomic profile of miRNAs was measured using NanoString nCounter® miRNA Expression Assays.
Project description:Pre-operative progesterone intervention has been shown to confer a survival benefit to breast cancer patients independent of their progesterone receptor (PR) status, raising a question about how progesterone affects the outcome of PR-negative cells. Here, we identify up-regulation of a Serum- and glucocorticoid-regulated kinase gene, SGK1 and an N-Myc Downstream Regulated Gene 1, NDRG1, along with down-regulation of miR-29a and miR-101-1 targeting 3’UTR region of SGK1, to differential extents in a PR dependent manner in breast cancer cells. We further demonstrate a novel dual-phase transcriptional and post-transcriptional regulation of SGK1 in response to progesterone leading to up-regulation of a tumor metastasis suppressor gene, NDRG1, mediated by a set of AP-1 network genes. The NDRG1 further inactivates a set of kinases impeding the invasion and migration of breast cancer cells. In summary, we propose a model for the mode of action of progesterone in breast cancer deciphering the molecular basis of a randomized clinical trial studying the effect of progesterone in breast cancer with a potential to improve the prognosis of breast cancer patients for receiving pre-operative progesterone treatment.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Host cells harbor various intrinsic mechanisms to restrict viral infections as a first line of antiviral defense. Viruses have evolved various countermeasures against these antiviral mechanisms. Here we show that N-Myc Downstream-Reguated Gene 1 (NDRG1) limits productive HCV infection by inhibiting viral assembly. Interestingly, HCV infection down-regulates NDRG1 protein and mRNA expression. Loss of NDRG1 increases the size and number of lipid droplets, which are the sites of HCV assembly. HCV suppresses NDRG1 expression by up-regulating MYC, which directly inhibits the transcription of NDRG1. Up-regulation of MYC also leads to reduced expression of NDRG1-specific kinase SGK1, resulting in markedly diminished phosphorylation of NDRG1. Knockdown of MYC during HCV infection rescues NDRG1 expression and phosphorylation, suggesting that MYC regulates NDRG1 at both transcriptional and post-translational levels. Overall, our results suggest that NDRG1 restricts HCV assembly by limiting lipid droplet formation. HCV counteracts this intrinsic antiviral mechanism by down-regulating NDRG1 via a MYC-dependent mechanism.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.