Dicer Knockout Research Articles

Cortical miR-709 links glutamatergic signaling to NREM sleep EEG slow waves in an activity-dependent manner.

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that have been implicated in a plethora of neuronal processes. Nevertheless, their role in regulating brain activity in the context of sleep has so far received little attention. To test their involvement, we deleted mature miRNAs in post-mitotic neurons at two developmental ages, i.e., in early adulthood using conditional Dicer knockout (cKO) mice and in adult mice using an inducible conditional Dicer cKO (icKO) line. In both models, electroencephalographic (EEG) activity was affected and the response to sleep deprivation (SD) altered; while the rapid-eye-movement sleep (REMS) rebound was compromised in both, the increase in EEG delta (1 to 4 Hz) power during non-REMS (NREMS) was smaller in cKO mice and larger in icKO mice compared to controls. We subsequently investigated the effects of SD on the forebrain miRNA transcriptome and found that the expression of 48 miRNAs was affected, and in particular that of the activity-dependent miR-709. In vivo inhibition of miR-709 in the brain increased EEG power during NREMS in the slow-delta (0.75 to 1.75 Hz) range, particularly after periods of prolonged wakefulness. Transcriptome analysis of primary cortical neurons in vitro revealed that miR-709 regulates genes involved in glutamatergic neurotransmission. A subset of these genes was also affected in the cortices of sleep-deprived, miR-709-inhibited mice. Our data implicate miRNAs in the regulation of EEG activity and indicate that miR-709 links neuronal activity during wakefulness to brain synchrony during sleep through the regulation of glutamatergic signaling.

Platelet-derived microRNAs play a pivotal role in cardiac remodeling after myocardial ischemia and reperfusion

Abstract Background Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of miRNAs, which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate, that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. Purpose The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. Methods and Results Combining an in vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (LSFM, PET&MRI, speckle-tracking echocardiography), in vitro immune cell migration assays and Next-generation deep sequencing analysis of platelet miRNA expression pattern in mice with a MK/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating LV remodeling after myocardial I/R following transient LCA ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial ischemia and reperfusion. Worsened cardiac remodeling after myocardial infarction (MI) in mice with a platelet-specific Dicer deletion (Dicer Pf4Δ/PfΔ4) resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at d28 post-MI. Altogether, these observations culminated in an impaired LV function and hampered long-term cardiac recovery after experimental MI and reperfusion therapy. Treatment with the P2Y12 antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in Dicer Pf4Δ/Pf4Δ mice. Conclusions The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial ischemia and reperfusion.

MiR-147 Represses NDUFA4, Inducing Mitochondrial Dysfunction and Tubular Damage in Cold Storage Kidney Transplantation.

Cold storage-associated transplantation (CST) injury occurs in renal transplant from deceased donors, the main organ source. The pathogenesis of CST injury remains poorly understood, and effective therapies are not available. This study has demonstrated an important role of microRNAs in CST injury and revealed the changes in microRNA expression profiles. Specifically, microRNA-147 (miR-147) is consistently elevated during CST injury in mice and in dysfunctional renal grafts in humans. Mechanistically, NDUFA4 (a key component of mitochondrial respiration complex) is identified as a direct target of miR-147. By repressing NDUFA4, miR-147 induces mitochondrial damage and renal tubular cell death. Blockade of miR-147 and overexpression of NDUFA4 reduce CST injury and improve graft function, unveiling miR-147 and NDUFA4 as new therapeutic targets in kidney transplantation. Kidney injury due to cold storage-associated transplantation (CST) is a major factor determining the outcome of renal transplant, for which the role and regulation of microRNAs remain largely unclear. The kidneys of proximal tubule Dicer (an enzyme for microRNA biogenesis) knockout mice and their wild-type littermates were subjected to CST to determine the function of microRNAs. Small RNA sequencing then profiled microRNA expression in mouse kidneys after CST. Anti-microRNA-147 (miR-147) and miR-147 mimic were used to examine the role of miR-147 in CST injury in mouse and renal tubular cell models. Knockout of Dicer from proximal tubules attenuated CST kidney injury in mice. RNA sequencing identified multiple microRNAs with differential expression in CST kidneys, among which miR-147 was induced consistently in mouse kidney transplants and in dysfunctional human kidney grafts. Anti-miR-147 protected against CST injury in mice and ameliorated mitochondrial dysfunction after ATP depletion injury in renal tubular cells in intro . Mechanistically, miR-147 was shown to target NDUFA4, a key component of the mitochondrial respiration complex. Silencing NDUFA4 aggravated renal tubular cell death, whereas overexpression of NDUFA4 prevented miR-147-induced cell death and mitochondrial dysfunction. Moreover, overexpression of NDUFA4 alleviated CST injury in mice. microRNAs, as a class of molecules, are pathogenic in CST injury and graft dysfunction. Specifically, miR-147 induced during CST represses NDUFA4, leading to mitochondrial damage and renal tubular cell death. These results unveil miR-147 and NDUFA4 as new therapeutic targets in kidney transplantation.

Abstract 3773: DeMixMir: deconvolution of microRNA sequencing data from heterogeneous tumor samples

Abstract Measuring tumor heterogeneity is a key issue in modern clinical oncology, with relevance to understanding cancer progression, resistance to therapy, and recurrence. Studies of the transcriptomic landscape in cancer have rapidly advanced in scale, enabled by novel methods and advancing technologies. One such novel method is TmS, a computational estimate of tumor-specific total messenger RNA content from heterogeneous tumor samples, which is calculated by combining genomic and transcriptomic deconvolutions. We have previously shown TmS as a promising pan-cancer biomarker for patient prognosis. Given the fruitful study of tumor cell total messenger RNA content, a logical extension is to investigate the utility of estimating tumor cell content of other RNA species. MicroRNAs (miRNAs) are small noncoding RNAs that regulate mRNA expression, and their dysregulation is a hallmark feature observed across cancers. A computational method to derive tumor cell total miRNA content from bulk sequencing data is especially needed since miRNAs cannot yet be reliably profiled at single cell resolution. Here we develop and benchmark DeMixMir, a new expansion of our reference-free transcriptomic deconvolutional model DeMixT, in order to recover tumor-specific miRNA proportions from mixed samples. For benchmarking, we generated an artificially mixed dataset of small RNA sequencing consisting of a total of 30 samples that were made by mixing HS-5 fibroblast cells with either wildtype or Dicer1 knockout HCT116 colorectal cancer cells. The tumor and fibroblast cell lines were mixed at 5 different proportions, to simulate a broad spectrum of tumor/nontumor cell mixing scenarios, with three independent replicates generated at each mixing ratio. DeMixMir demonstrated high accuracy in estimating the tumor-specific miRNA proportions in both the wildtype and the Dicer1 knockout mixtures. We further calculated tumor cell total miRNA content using DeMixMir output. Our proof-of-concept study suggests estimating tumor cell total miRNA content across tumor tissues at scale is feasible and likely valuable for advancing understanding of the complex roles miRNAs play in the cancer ecosystem. Citation Format: Matthew Montierth, Kinga Nemeth, Xinghua Tao, George Calin, Wenyi Wang. DeMixMir: deconvolution of microRNA sequencing data from heterogeneous tumor samples. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3773.

Hepatic Glucose Metabolism Disorder Induced by Adipose Tissue-Derived miR-548ag via DPP4 Upregulation.

The present study aimed to explore the molecular mechanism underlying the regulation of glucose metabolism by miR-548ag. For the first time, we found that miR-548ag expression was elevated in the abdominal adipose tissue and serum of subjects with obesity and type 2 diabetes mellitus (T2DM). The conditional knockout of adipose tissue Dicer notably reduced the expression and content of miR-548ag in mouse adipose tissue, serum, and liver tissue. The combined use of RNAseq, an miRNA target gene prediction software, and the dual luciferase reporter assay confirmed that miR-548ag exerts a targeted regulatory effect on DNMT3B and DPP4. miR-548ag and DPP4 expression was increased in the adipose tissue, serum, and liver tissue of diet-induced obese mice, while DNMT3B expression was decreased. It was subsequently confirmed both in vitro and in vivo that adipose tissue-derived miR-548ag impaired glucose tolerance and insulin sensitivity by inhibiting DNMT3B and upregulating DPP4. Moreover, miR-548ag inhibitors significantly improved the adverse metabolic phenotype in both obese mice and db/db mice. These results revealed that the expression of the adipose tissue-derived miR-548ag increased in obese subjects, and that this could upregulate the expression of DPP4 by targeting DNMT3B, ultimately leading to glucose metabolism disorder. Therefore, miR-548ag could be utilized as a potential target in the treatment of T2DM.

Dicer prevents activation of the type I interferon pathway in lipid-loaded macrophages

Abstract Background The expression of the endonuclease Dicer in macrophages decreases atherosclerosis and necrotic core formation by producing microRNAs such as miR-10a. This effect of Dicer is associated with enhanced mitochondrial respiration in lipid-loaded macrophages. However, the mechanism by which Dicer-dependent production of miRNAs in lipid-loaded macrophages regulates mitochondrial function is unclear. Purpose We aimed to determine the effect of Dicer on lipid-loaded macrophages in the context of atherosclerosis. Methods Mice with a myeloid cell-specific knockout of Dicer (Lys-Cre/Dicerflox/flox/Apoe−/− mice [M-Dicer−/−]) and control mice (Lys-Cre/DicerWT/WT/Apoe−/− mice [M-Dicer+/+]) were fed a high-fat diet (HFD) for 24 weeks. The oxygen consumption rate (OCR) in aortic arch plaques was studied ex vivo by Seahorse Flux XF 24 Analyzer. Bone marrow-derived macrophages (BMDMs) were stimulated with oxLDL (100 μg/mL) for 72 h. Lipid-loaded macrophages were used for proteomic analysis by mass spectrometry. RIP-prime- seq was performed in tAgo2 immunoprecipitates (IP) from Dicer+/+ and Dicer−/− BMDMs. The miRNA expression profile was determined in lipid-loaded BMDMs by NanoString technology. Stat1 phosphorylation was determined in lipid-loaded macrophages by Jess automated western blot system (ProteinSimple). Result The OCR in aortic arch tissue with plaques was higher than in those without plaques in M-Dicer+/+ mice after 24 weeks of HFD feeding. Dicer knockout in macrophages decreased the OCR in aortic arch tissues with plaques but not in aortic arch tissues without plaques (n=3–4, p<0.05). The proteomic analysis of oxLDL-treated BMDMs indicated that Dicer knockout activated the type I interferon signaling pathway by up-regulating the expression of STAT1/2 and interferon-stimulated genes, such as ISG15. Proteins related to mitochondrial DNA were upregulated (e.g., Dnmt3a) or downregulated (e.g., Tfam) by Dicer knockout (n=5, adj.p<0.05). RIP-prime-seq from BMDMs showed that 1376 transcripts were significantly enriched in the tAgo IP from Dicer+/+ compared with that from Dicer−/− BMDMs (n=3–5, adj.p<0.05) including Dnmt3a and Stat2. STAT1 phosphorylation was increased in Dicer−/− compared with Dicer+/+ BMDMs. Among the 299 miRNAs downregulated by Dicer knockout in lipid-loaded macrophages (n=6, adj.p<0.05), miR-29 and miR-30 have highly conserved binding sites (predicted by TargetScan) for Dnmt3a. Conclusion Our results indicate that Dicer expression in lipid-loaded macrophages limits Stat1/2-driven type I interferon response due to mitochondrial damage. This effect may be mediated by the suppression of Dnmt3a by miRNAs such as miR-29 and miR-30. This suggests that targeting Dnmt3a-mediated mitochondrial damage in lipid-loaded macrophages by miRNAs may be therapeutic strategy to limit atherosclerosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG

Dicer deficiency impairs proliferation but potentiates anti-tumoral effect of macrophages in glioblastoma.

Glioblastoma is a lethal primary brain tumor with abundant immune-suppressive glioblastoma-associated macrophage (GAM) infiltration. Skewing immune suppressive GAMs towards an immune-activating phenotype represents a promising immunotherapeutic strategy against glioblastoma. Herein, we reported that genetic deletion of miRNA-processing enzyme Dicer in macrophages inhibited the growth of GL261 murine glioblastoma xenografts and prolonged survival of tumor-bearing mice. Single cell RNA sequencing (scRNA-seq) of the tumor-infiltrating immune cells revealed that Dicer deletion in macrophages reduced the proportion of cell-cycling GAM cluster and reprogramed the remaining GAMs towards a proinflammatory activation state (enhanced phagocytotic and IFN-producing signature). Dicer-deficient GAMs showed reduced level of cyclin-dependent kinases (CDK1 and CDK2) and increased expression of CDK inhibitor p27 Kip1, thus manifesting impaired proliferation. Dicer knockout enhanced phagocytotic activity of GAMs to eliminate GL261 tumor cells. Increased proinflammatory GAM clusters in macrophage Dicer-deficient mice actively interacted with tumor-infiltrating T cells and NK cells through TNF paracrine signaling to create a pro-inflammatory immune microenvironment for tumor cell elimination. Our work identifies the role of Dicer deletion in macrophages in generating an immune-activating microenvironment, which could be further developed as a potential immunotherapeutic strategy against glioblastoma.

The Effect of Dicer Knockout on RNA Interference Using Various Dicer Substrate Small Interfering RNA (DsiRNA) Structures.

Small interfering RNAs (siRNAs) are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonuclease Dicer. Dicer-substrate small interfering RNAs (DsiRNAs) are an alternative to conventional 21-mer siRNAs, with an increased effectiveness of up to 100-fold compared to traditional 21-mer designs. DsiRNAs have a novel asymmetric design that allows them to be processed by Dicer into the desired conventional siRNAs. DsiRNAs are a useful tool for sequence-specific gene silencing, but the molecular mechanism underlying their increased efficacy is not precisely understood. In this study, to gain a deeper understanding of Dicer function in DsiRNAs, we designed nicked DsiRNAs with and without tetra-loops to target a specific mRNA sequence, established a Dicer knockout in the HCT116 cell line, and analyzed the efficacy of various DsiRNAs on RNAi-mediated gene silencing activity. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. We demonstrated that tetra-looped DsiRNAs exhibited increased efficacy for gene silencing, which was mediated by Dicer protein. Thus, this study improves our understanding of Dicer function, a key component of RNAi silencing, which will inform RNAi research and applications.

Abstract WP250: Exosomes Derived From Different Regional Cerebral Endothelial Cells Have Distinct Effects On Adult Rodent Neurogenesis

Introduction: Exosomes mediate intercellular communication. We tested the hypothesis that exosomes derived from different area cerebral endothelial cells (CECs) have distinct effects on adult neurogenesis. Methods and Results: Exosomes were isolated from CECs of cortical (Cor-CEC-exo) and subventricular zone (SVZ-CEC-exo) vessels of non-stroke and stroke rats (n=4/group). Neural stem cells (NSCs) from the non-stroke SVZ were treated with CEC-exo. Proliferation and differentiation of NSCs were measured by BrdU + cells and Tuj1 + neuroblasts. There were no significant differences of size distribution and marker proteins among studied exosomes. Treatment of NSCs with non-stroke Cor-CEC-exo did not significantly change NSC proliferation (35%± 2% vs 31%±2% in control) and neuronal differentiation (5%±2% vs 5%± 0.1% in control). However, treatment of NSCs with non-stoke SVZ-CEC-exo significantly (p<0.05) increased BrdU + (42%± 2%) and Tuj1 + cells (10%± 2%). These data suggest that non-stroke Cor-CEC-exo, but not SVZ-CEC-exo, do not affect neurogenesis. RT-PCR analysis showed that Cor-CEC-exo were enriched in selective miRNAs including miR-203 that is known to negatively regulate NSC proliferation. To study whether miRNAs are involved in the effect of CEC-exo on neurogenesis, we isolated NSCs in which miRNA biogenesis gene, Dicer, was ablated. Compared to wild-type (WT) NSCs, ablation of Dicer led to significant reduction of proliferation (16%±1%) and neuronal differentiation (1%±0.1%). Treatment of Dicer knockout NSCs with Cor- or SVZ-CEC-exo significantly increased BrdU+ (25%±2% or 34%±2%) and TuJ1+ cells (3%±0.1% or 5%±0.1%), suggesting that both Cor- and SVZ-CEC-exo can rescue Dicer knockout impaired neurogenesis. Moreover, both stroke Cor- and SVZ-CEC-exo significantly augmented non-stroke WT NSC proliferation (41%±3% and 51%±4%) and differentiation (9%±0.2% and 16%±0.3). Conclusion: Our data demonstrate that under non-stroke condition, Cor-CEC-exo do not promote neurogenesis, while SVZ-CEC-exo enhance neurogenesis. After stroke, both Cor-and SVZ-CEC-exo augment neurogenesis. The effect of CEC-exo on neurogenesis is mediated by miRNAs. This study provides new insight into CEC-exo in mediating neurogenesis.

Molecular Mechanisms of Parathyroid Disorders in Chronic Kidney Disease.

Secondary hyperparathyroidism (SHP) is a common complication of chronic kidney disease (CKD) that induces morbidity and mortality in patients. How CKD stimulates the parathyroid to increase parathyroid hormone (PTH) secretion, gene expression and cell proliferation remains an open question. In experimental SHP, the increased PTH gene expression is post-transcriptional and mediated by PTH mRNA–protein interactions that promote PTH mRNA stability. These interactions are orchestrated by the isomerase Pin1. Pin1 participates in conformational change-based regulation of target proteins, including mRNA-binding proteins. In SHP, Pin1 isomerase activity is decreased, and thus, the Pin1 target and PTH mRNA destabilizing protein KSRP fails to bind PTH mRNA, increasing PTH mRNA stability and levels. An additional level of post-transcriptional regulation is mediated by microRNA (miRNA). Mice with parathyroid-specific knockout of Dicer, which facilitates the final step in miRNA maturation, lack parathyroid miRNAs but have normal PTH and calcium levels. Surprisingly, these mice fail to increase serum PTH in response to hypocalcemia or uremia, indicating a role for miRNAs in parathyroid stimulation. SHP often leads to parathyroid hyperplasia. Reduced expressions of parathyroid regulating receptors, activation of transforming growth factor α-epidermal growth factor receptor, cyclooxygenase 2-prostaglandin E2 and mTOR signaling all contribute to the enhanced parathyroid cell proliferation. Inhibition of mTOR by rapamycin prevents and corrects the increased parathyroid cell proliferation of SHP. This review summarizes the current knowledge on the mechanisms that stimulate the parathyroid cell at multiple levels in SHP.

Ablation of microRNAs in VIP+ interneurons impairs olfactory discrimination and decreases neural activity in the olfactory bulb.

MicroRNAs (miRNAs) are abundantly expressed in vasoactive intestinal peptide expressing (VIP+ ) interneurons and are indispensable for their functional maintenance and survival. Here, we blocked miRNA biogenesis in postmitotic VIP+ interneurons in mice by selectively ablating Dicer, an enzyme essential for miRNA maturation, to study whether ablation of VIP+ miRNA affects olfactory function and neural activity in olfactory centres such as the olfactory bulb, which contains a large number of VIP+ interneurons. A go/no-go odour discrimination task and a food-seeking test were used to assess olfactory discrimination and olfactory detection. In vivo electrophysiological techniques were used to record single units and local field potentials. Olfactory detection and olfactory discrimination behaviours were impaired in VIP+ -specific Dicer-knockout mice. In vivo electrophysiological recordings in awake, head-fixed mice showed that both spontaneous and odour-evoked firing rates were decreased in mitral/tufted cells in knockout mice. The power of ongoing and odour-evoked beta local field potentials response of the olfactory bulb and anterior piriform cortex were dramatically decreased. Furthermore, the coherence of theta oscillations between the olfactory bulb and anterior piriform cortex was decreased. Importantly, Dicer knockout restricted to olfactory bulb VIP+ interneurons recapitulated the behavioural and electrophysiological results of the global knockout. VIP+ miRNAs are an important factor in sensory processing, affecting olfactory function and olfactory neural activity.

MicroRNA‐mRNA genetic networks in refractive eye development and myopia

AbstractPurposeMyopia is a complex disease controlled by both environmental and genetic factors. Recent genetic studies suggested that the development of myopia is controlled by numerous coding genes. However, the contribution of non‐coding genes, such as microRNAs, is poorly understood. The purpose of this study was to investigate the role of microRNAs in refractive eye development and the development of myopia.MethodsDifferential expression of both microRNAs and mRNAs was analyzed in the retina, RPE, choroid, and sclera of C57BL mice upon induction of experimental myopia with −20 D lenses using RNA‐seq and microRNA‐mRNA genetic networks were reconstructed. We also explored the impact of microRNAs on signaling pathways and evaluated the role of microRNAs in refractive eye development in vivo using conditional Dicer1 knockout mice.ResultsGene expression profiling revealed large number of differential mRNAs and microRNAs in the retina and RPE/choroid/sclera complexes. MicroRNAs were involved in the regulation of several key canonical pathways and biological processes previously implicated in refractive eye development. We then compared visual acuity, contrast sensitivity, and refractive errors in Dicer1 knockouts (Dicer1−/−) and wild‐type littermates (Dicer1+/+) at baseline. We found that loss of miRNAs in the postnatal eyes resulted in a large hyperopic shift in refractive error (+16.3 ± 3.6 D in Dicer1 knockouts versus +4.7 ± 3.0 D in wild‐type animals, p < 1.44 × 10−4), which was accompanied by a small reduction in visual acuity (0.360 ± 0.006 cpd in Dicer1−/− versus 0.403 ± 0.002 cpd in Dicer1+/+, p < 1.16 × 10−7) and contrast sensitivity (12.01 ± 0.27 in Dicer1−/− versus 13.65 ± 0.40 in Dicer1+/+, p < 1.91 × 10−5). Loss of miRNAs also caused a statistically significant increase in susceptibility to form‐deprivation myopia in Dicer1 knockouts compared to wild‐type animals (−19.1 ± 6.8 D in Dicer1−/− versus −9.7 ± 2.7 D in Dicer1+/+, p < 1.51 × 10−4).ConclusionsOur data suggest that microRNAs play significant role in refractive eye development and the development of myopia via modulation of several canonical signaling pathways. MicroRNAs appear to be integrated in the signaling cascade involved in the optical‐defocus‐regulated refractive eye development at multiple levels.

Dicer-mediated miRNA processing is not involved in controlling muscle mass during muscle atrophy

Muscle atrophy occurs in a variety of physiological and pathological conditions. Specific molecular networks that govern protein synthesis and degradation play important roles in controlling muscle mass under diverse catabolic states. MicroRNAs (miRNAs) were previously found to be regulators of protein synthesis and degradation, and their expressions in skeletal muscle were altered in muscle wasting conditions. However, functional roles of miRNAs in muscle atrophy are poorly understood. In this study, we generated tamoxifen-inducible Dicer knockout (iDicer KO) mice and subjected them to 2 weeks of single hindlimb denervation. The expression of Dicer mRNA was significantly reduced in muscle of the iDicer KO mice compared to that of WT mice. The loss of Dicer moderately reduced levels of muscle-enriched miRNAs, miR-1, miR-133a and miR-206 in both innervated and denervated muscles of the iDicer KO mice. We also found that the extent of denervation-induced muscle atrophy as well as changes of signaling molecules related to protein synthesis/degradation pathways in the iDicer KO mice were comparable to these in WT mice. Taken together, Dicer knockout in adult skeletal muscle did not affect denervation-induced muscle atrophy.

Generation of Self-Inhibitory Recombinant Viral Hemorrhagic Septicemia Virus (VHSV) by Insertion of Viral P Gene-Targeting Artificial MicroRNA into Viral Genome and Effect of Dicer Gene Knockout on the Recombinant VHSV Replication.

To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P. The replication of rVHSV-A-amiR-C in EPC-ΔDicer cells was not different from that in control EPC cells, while the copy number of rVHSV-A-amiR-P was increasingly risen up in EPC-ΔDicer cells compared to that in control EPC cells, and the final viral titer of rVHSV-A-amiR-P was enhanced by culture in EPC-ΔDicer cells. These results indicate that VHSV can be attenuated by the equipment of self-mRNA-targeting microRNA sequence in the genome, and the titer of artificial miRNA-expressing attenuated recombinant VHSVs can be enhanced by the knockout of Dicer gene in EPC cells.

Widespread formation of double-stranded RNAs in testis.

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes—again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.

Dicer Deletion Leads to Different Antiviral Responses in Mouse Embryonic Stem Cells

Interferon (IFN) response is the primary antiviral mechanism presumably developed in most, if not all, types of mammalian cells. However, recent studies have demonstrated that embryonic stem cells (ESCs) from both human and mouse are deficient in expressing type I IFN. It is proposed that ESCs may use RNA interference (RNAi) as an alternative antiviral system, which is the major antiviral mechanism in invertebrates but not normally operational in mammalian cells. RNAi uses short interfering RNA (siRNA) derived from viral RNA to limit viral replication. Since Dicer is a key enzyme for siRNA biogenesis, Dicer knockout ESCs (D-/-ESCs) could be used as a loss of function model to determine antiviral RNAi activity in ESCs. In this study, we compared the infectivity of Chikungunya virus (ChikV) and La Crosse virus (LaCV) in normal mouse ESCs and D-/-ESCs. It is expected that ESCs would be more susceptible to viral infection than D-/-ESCs if RNAi is functional ESCs. This is true in the case of LaCV infection, but surprisingly, D-/-ESCs is more resistant to ChikV infection than ESCs, indicating that other antiviral pathways may also be involved. These results could be explained by our recent findings that D-/-ESCs have gained the ability to express IFNβ, which is otherwise deficient in wild-type ESCs. Furthermore, D-/-ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), which also plays an important role in antiviral response. Our results demonstrated that inhibiting PKR increased the infectivity of both viruses in D-/-ESCs and ESCs. Incubation of ESCs with neutralizing antibodies against IFNβ did not have an effect on viral infectivity in ESCs as expected. However, neutralizing antibody treatment increased the infectivity of ChikV, but not LaCV, in D-/-ESCs. Based on these findings, we can conclude that PKR can provide antiviral activity to both ESCs and D-/-ESCs, but IFNβ is only produced in D-/-ESCs and shows its effect in a virus-dependent manner. We are currently investigating the molecular mechanisms underlying this interesting finding.

Dicer represses the interferon response and the double-stranded RNA-activated protein kinase pathway in mouse embryonic stem cells

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D−/−ESCs) as expected. Surprisingly, D−/−ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D−/−ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D−/−ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D−/−ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D−/−ESCs. Treatment of D−/−ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs.

Gonadal white adipose tissue-derived exosomal MiR-222 promotes obesity-associated insulin resistance.

In this study, we investigated the role of serum exosomal miR-222 in obesity-related insulin resistance. Bioinformatics analyses showed that miR-222 levels were significantly upregulated in the white adipose tissue of obese patients with insulin resistance (GSE25402 dataset) and in serum samples from type 2 diabetes mellitus (T2DM) patients (GSE90028 dataset). Moreover, analysis of miRNA expression in adipose tissue-specific Dicer knockout mice (GitHub dataset) and diabetic model mice (GSE81976 and GSE85101 datasets), gonadal white adipose tissue (gWAT) was the main source of serum exosomal miR-222. MiR-222 levels were significantly elevated in the serum, serum exosomes and gWAT of mice fed a high-fat diet (HFD), and there was a corresponding downregulation of IRS1 and phospho-AKT levels in their liver and skeletal muscle tissues, which correlated with impaired insulin sensitivity and glucose intolerance. These effects were abrogated by surgically removing the gWAT from the HFD-fed mice. Thus, gWAT-derived serum exosomal miR-222 appears to promote insulin resistance in the liver and skeletal muscle of HFD-fed obese mice by suppressing IRS1 expression.

Intestinal Lipid Metabolism Genes Regulated by miRNAs.

MicroRNAs (miRNAs) crucial roles in translation repression and post-transcriptional adjustments contribute to regulate intestinal lipid metabolism. Even though their actions in different metabolic tissues have been elucidated, their intestinal activity is yet unclear. We aimed to investigate intestinal miRNA-regulated lipid metabolism-related genes, by creating an intestinal-specific Dicer1 knockout (Int-Dicer1 KO) mouse model, with a depletion of microRNAs in enterocytes. The levels of 83 cholesterol and lipoprotein metabolism-related genes were assessed in the intestinal mucosa of Int-Dicer1 KO and Wild Type C57BL/6 (WT) littermates mice at baseline and 2 h after an oral lipid challenge. Among the 18 genes selected for further validation, Hmgcs2, Acat1 and Olr1 were found to be strong candidates to be modulated by miRNAs in enterocytes and intestinal organoids. Moreover, we report that intestinal miRNAs contribute to the regulation of intestinal epithelial differentiation. Twenty-nine common miRNAs found in the intestines were analyzed for their potential to target any of the three candidate genes found and validated by miRNA-transfection assays in Caco-2 cells. MiR-31-5p, miR-99b-5p, miR-200a-5p, miR-200b-5p and miR-425-5p are major regulators of these lipid metabolism-related genes. Our data provide new evidence on the potential of intestinal miRNAs as therapeutic targets in lipid metabolism-associated pathologies.

DICER regulates the expression of major satellite repeat transcripts and meiotic chromosome segregation during spermatogenesis

Constitutive heterochromatin at the pericentric regions of chromosomes undergoes dynamic changes in its epigenetic and spatial organization during spermatogenesis. Accurate control of pericentric heterochromatin is required for meiotic cell divisions and production of fertile and epigenetically intact spermatozoa. In this study, we demonstrate that pericentric heterochromatin is expressed during mouse spermatogenesis to produce major satellite repeat (MSR) transcripts. We show that the endonuclease DICER localizes to the pericentric heterochromatin in the testis. Furthermore, DICER forms complexes with MSR transcripts, and their processing into small RNAs is compromised in Dicer1 knockout mice leading to an elevated level of MSR transcripts in meiotic cells. We also show that defective MSR forward transcript processing in Dicer1 cKO germ cells is accompanied with reduced recruitment of SUV39H2 and H3K9me3 to the pericentric heterochromatin and meiotic chromosome missegregation. Altogether, our results indicate that the physiological role of DICER in maintenance of male fertility extends to the regulation of pericentric heterochromatin through direct targeting of MSR transcripts.