ABSTRACT
Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy.
Subject(s)
Breast Neoplasms/genetics , Colonic Neoplasms/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , RNA-Binding Proteins/genetics , Amino Acid Sequence , Breast Neoplasms/pathology , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Colonic Neoplasms/pathology , Female , Humans , Molecular Sequence Data , Neoplasm Invasiveness , RNA-Binding Proteins/chemistry , Transcription, GeneticABSTRACT
Several authors have described methods to track perfusion and cardiac surgical morbidity and mortality as well as perfusion accidents. There is currently not a standard definition of a perfusion accident nor is there a standard reporting threshold for events which do not directly cause known morbidity. We propose the term non-routine events (NREs) instead of accidents, and provide a working definition and reporting threshold for such. This paper describes the program which we developed to track perfusion NREs within the Cardiovascular Program at Children's Hospital, Boston. NREs are categorized by type (technique, equipment, or patient-related) and bypass period (pre-cardiopulmonary bypass, bypass, or post-cardiopulmonary). NRE outcomes are also classified by the level of discussion or change in perfusion practice after multidisciplinary review. We have documented during a 44 month interval that 42% (29/69) of reported NREs occur during the bypass period and are equipment related and thus, efforts to improve practice should focus there. We have also seen a generally decreasing incidence of NREs requiring either a change in perfusion practice or a new protocol during this time period. We believe that our regular multidisciplinary meetings to discuss NREs have increased awareness among the entire team about potential problems in the program and that intuitively, it has improved patient safety.
Subject(s)
Extracorporeal Circulation/standards , Mandatory Reporting , Quality Assurance, Health Care/methods , Quality Assurance, Health Care/organization & administration , Risk Management/methods , Risk Management/organization & administration , Boston , Practice Guidelines as TopicABSTRACT
The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.
Subject(s)
MicroRNAs/metabolism , Proteins/genetics , RNA, Messenger/metabolism , Ribonuclease III/metabolism , 5' Untranslated Regions , Cells, Cultured , HeLa Cells , Humans , Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA-Binding Proteins , Ribonuclease III/genetics , TransfectionABSTRACT
The developmentally regulated RNA-binding protein Lin28 blocks processing of let-7 family microRNAs (miRNAs) in embryonic cells. The molecular basis for this selective miRNA processing block is unknown. Here we find that Lin28 selectively binds the terminal loop region of let-7 precursors in vitro and that the loop mediates miRNA processing inhibition in vivo. Additionally, we identify the domains of Lin28 required for this inhibition. These findings establish a regulatory role for the terminal loop of precursors in miRNA maturation and provide insight into the mechanism by which Lin28 negatively regulates let-7 processing.
Subject(s)
Gene Expression Regulation , MicroRNAs/metabolism , RNA-Binding Proteins/chemistry , Base Sequence , Cell Line, Tumor , Humans , Kinetics , MicroRNAs/genetics , Models, Biological , Molecular Sequence Data , Mutagenesis , Protein Binding , Protein Structure, Tertiary , RNA Interference , Sequence Homology, Nucleic AcidABSTRACT
Bivalirudin, a direct thrombin inhibitor, has recently emerged as a promising option for anti-coagulation during cardiopulmonary bypass in patients who cannot receive heparin. There is limited experience with the use of bivalirudin in children. We present the case of a child with heparin-induced thrombocytopenia with thrombosis (HIT Type II) who underwent successful orthotopic cardiac transplantation using bivalirudin as the primary anti-coagulant for cardiopulmonary bypass.
