Unknown,Transcriptomics,Genomics,Proteomics

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Nanostring microRNA microarry analysis of p53 and Mdm2 knockout mouse hearts compared to vehicle-injected negative controls

ABSTRACT: Adult cardiomyocytes (CM) are terminally differentiated cells with minimal regenerative capacity, making cardiac tissue particularly vulnerable to injury. Thus, defining the roadblocks responsible for adult CM cell cycle arrest lies at the core of developing therapies to regenerate myocyte loss following injurious events such as myocardial infarction. We have previously shown that inactivating the p53/Mdm2 tumor suppressor circuitry, specifically in the heart (using the Cre-loxP recombination system of bacteriophage P1), can allow differentiated CMs to regain proliferative capacity, through an upregulation of factors involved in cell cycle re-entry. These factors are repressed in quiescent CMs, in part through the action of microRNAs (miRNAs). Notably, knockout of either p53 or Mdm2 individually was insufficient to promote CM proliferation. Therefore, we hypothesized that inactivation of p53/Mdm2-regulated miRNAs could promote the expression of cell cycle activators and induce proliferation of adult murine CMs. To identify miRNAs regulated by both p53 and Mdm2, total miRNA expression profiles from cardiac specific p53/Mdm2 double knockout (DKO) mouse hearts were compared with those from cardiac-specific single knockouts (p53KO and Mdm2KO), and vehicle-injected controls using the Nanostring nCounter mouse miRNA expression assay. This revealed a profile of 11 significantly downregulated miRNAs in the proliferative DKO hearts (versus vehicle-injected control), that were enriched for mRNA targets involved in cell cycle regulation. In vitro studies have demonstrated that knockdown of these 11 miRNAs in neonatal rat cardiomyocytes can increase the occurrence of cytokinetic events. Ultimately, we aim to inject antagomirs targeting these miRNAs into animals post-myocardial infarction to determine the effect of p53/Mdm2-regulated miRNAs on heart function and CM proliferation in vivo.

ORGANISM(S): Mus musculus

SUBMITTER: Shanna Stanley-Hasnain

PROVIDER: E-MTAB-5531 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion: Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. Livers from C57Bl/6 mice were isolated at defined ages: embryonic day 15.5 (n=3; mixed gender), 2 weeks (n=3; male), 3 weeks (n=3, male) and 7 weeks (n=3; male). Differential miRNA expression was assessed using the nCounter Mouse miRNA Expression Assay Kit (nanoString).

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Nanostring microRNA microarry analysis of p53 and Mdm2 knockout mouse hearts compared to vehicle-injected negative controls

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