Dicer Deficiency Research Articles

Abstract 5756: The MicroRNA-17–92 Cluster Exhibits a Cell Intrinsic Anti-angiogenic Function in Endothelial Cells

MicroRNAs (miRs) are endogenously expressed small noncoding RNA molecules that are able to regulate gene expression on the posttranscriptional level by binding to the mRNAs of their target genes, thus inducing inhibition of translation or mRNA degradation. The miR-17–92 cluster (encoding miR-17, -18a, -19a, -20a, -19b and miR-92a) was shown to augment tumor angiogenesis if overexpressed in tumor cells. Whereas miR-92a was recently identified as negative regulator of vessel growth, the individual functions of the other members of the miR-17–92 cluster in endothelial cells is less clear and only a combination of miR-17, -18a and -20a was shown to partially rescue the defective angiogenesis under conditions of Dicer deficiency. To investigate the involvement of the individual members of the miR-17–92 cluster in angiogenesis, human umbilical vein endothelial cells (HUVECs) were transfected with precursor molecules for miR-17, -18a, -19a and -20a. Overexpression of the different members significantly inhibited 3D spheroid sprouting (Pre-17:10±3%,-18a: 28±5%,-19a: 45±4%,-20a: 33±8% of control) and impaired the assembly of capillary networks in vitro. Conversely, inhibition of miR-17 (142±15% of control), miR-18a (152±6% of control) and miR-20a (153±9% of control) augmented spheroid sprout formation. Next, we addressed the in vivo relevance by using antagomirs to block the individual miRs. Antagomir-17 (which also blocks miR-20a) significantly increased the number of lectin-positive vessels in matrigel plugs (190±26% of control), whereas antagomirs, which specifically affect miR-18a and miR-19a were less effective. Antagomir-17 additionally improved blood flow after hind limb ischemia (217±24% of control) and increased tumor growth (140±14% of control). To identify direct targets of miR-17, we combined microarray gene expression analysis with target prediction. Among others, Janus kinase 1 (Jak1) was significantly downregulated on mRNA (38±5% of control) and protein (41±14% of control) level by miR-17. In summary, we show that the individual members of the miR-17–92 clusters exhibit a cell intrinsic anti-angiogenic activity in endothelial cells. Inhibition of miR-17 efficiently increased neovascularization and tumor growth in vivo.

Abstract 5321: Dicer Depletion in Adult Hearts Induces Oxidative Stress, Mitochondrial Dysfunction and Causes Rapid Loss of Cardiac Function

Introduction. Dicer, an RNAse III endonuclease, plays a key role in the processing of miRNA into their functional mature form. A number of recent reports point towards a central role of miRNA in cardiac development and function. However, the short-term effects of dicer deficiency on the function of the adult heart and underlying mechanisms remain to be elucidated. Oxidative stress and dysregulated mitochondrial function are known to play a major role in acute cardiac pathologies. Hypothesis . Arrest of miR maturation by heart-specific dicer knock-out in adult animals result in rapid loss of cardiac function caused by mitochondrial dysfunction and oxidative stress. Methods . Mice homozygous for Dicer-floxed alleles (Dicer fl/fl ) and transgenic Myh6-cre/Esr1 mice were crossed to generate double-transgenic (Myh6-cre/Esr1-Dicer fl/fl ) mice. At 8 weeks of age Myh6-cre/Esr1-Dicer fl/fl mice were treated with vehicle or tamoxifen (tam; 20 mg/kg per day) daily ip injections for five consecutive days. To determine the acute functional consequences of Dicer depletion, heart function was determined using gated cardiac MRI and echocardiography. Myocardial miR profiling was performed using a bead array system. Candidate miRs were verified using quantitative PCR. Results . Tam injection for 5 days effectively depleted Dicer protein in myocardium. miRNA array data show significant (p< 0.05, n=3) decrease in the levels of a number of abundant mature miRNA including miR-1, miR-133 a&b. Following 5 days of Tam injection, a significant (p< 0.05, n=5) decrease in ejection fraction (32±9%↓), stroke volume (23±7%↓) and cardiac output (28.3±6%↓) was noted in Dicer −/− compared to Dicer +/+ mice. Studies with isolated mitochondria showed a significant decrease in energy dependent pore opening and increase in uncoupled mitochondria in the heart of Dicer −/− mice. Such compromise in mitochondrial function was associated with structural disintegration and increased (30±7.5%↑; p<0.05, n=3) tissue oxidative stress. Conclusions . This study provides first evidence demonstrating that depletion of dicer in adult mice results in mitochondrial dysfunction and oxidative stress in the myocardium which is followed by acute loss of cardiac functions.

Dicer Ablation Affects Antibody Diversity and Cell Survival in the B Lymphocyte Lineage

To explore the role of Dicer-dependent control mechanisms in B lymphocyte development, we ablated this enzyme in early B cell progenitors. This resulted in a developmental block at the pro- to pre-B cell transition. Gene-expression profiling revealed a miR-17 approximately 92 signature in the 3'UTRs of genes upregulated in Dicer-deficient pro-B cells; a top miR-17 approximately 92 target, the proapoptotic molecule Bim, was highly upregulated. Accordingly, B cell development could be partially rescued by ablation of Bim or transgenic expression of the prosurvival protein Bcl-2. This allowed us to assess the impact of Dicer deficiency on the V(D)J recombination program in developing B cells. We found intact Ig gene rearrangements in immunoglobulin heavy (IgH) and kappa chain loci, but increased sterile transcription and usage of D(H) elements of the DSP family in IgH, and increased N sequence addition in Igkappa due to deregulated transcription of the terminal deoxynucleotidyl transferase gene.