Loss Of Dicer1 Research Articles

DICER1 mutations in primary central nervous system tumors: new insights into histologies, mutations, and prognosis.

We sought to characterize clinical outcomes for adult and pediatric patients with primary CNS tumors harboring DICER1 mutations or loss of DICER1. We conducted a retrospective cohort study of 98 patients who were treated between 1995 and 2020 for primary CNS tumors containing DICER1 mutations or loss of DICER1 on chromosome 14q, identified by targeted next generation sequencing. Kaplan-Meier plots and log rank tests were used to analyze survival. Cox proportional-hazards model was used for univariate and multivariable analyses for all-cause mortality (ACM). Within our cohort, the most common malignancies were grade 3/4 glioma (61%), grade 1/2 glioma (17%), and CNS sarcoma (6%). Sarcoma and non-glioma histologies, and tumors with biallelic DICER1 mutations or deletions were common in the pediatric population. Mutations occurred throughout DICER1, including missense mutations in the DexD/H-box helicase, DUF283, RNaseIIIa, and RNaseIIIb domains. For patients with grade 3/4 glioma, MGMT methylation (Hazard ratio [HR] 0.35, 95% Confidence Interval [CI] 0.16-0.73, p = 0.005), IDH1 R132 mutation (HR 0.11, 95% CI 0.03-0.41, p = 0.001), and missense mutation in the DexD/H-box helicase domain (HR 0.06, 95% CI 0.01-0.38, p = 0.003) were independently associated with longer time to ACM on multivariable analyses. DICER1 mutations or loss of DICER1 occur in diverse primary CNS tumors, including previously unrecognized grade 3/4 gliomas as the most common histology. While prior studies have described RNaseIIIb hotspot mutations, we document novel mutations in additional DICER1 functional domains. Within the grade 3/4 glioma cohort, missense mutation in the DexD/H-box helicase domain was associated with prolonged survival.

Genetic context of oncogenic drivers dictates vascular sarcoma development in aP2-Cre mice.

Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2‐Cre‐mediated biallelic loss of Dicer1 or conditional activation of Kras G12D with Cdkn2a loss that histologically and genetically resemble human tumors. In the present study, we found that DICER1 functions as a potent tumor suppressor and its deletion, in combination with either KRASG12D expression or Cdkn2a loss, is associated with angiosarcoma development. Independent of the genetic driver, the mTOR pathway was activated in all murine angiosarcoma models. Direct activation of the mTOR pathway by conditional deletion of Tsc1 with aP2‐Cre resulted in tumors that resemble intermediate grade human kaposiform hemangioendotheliomas, indicating that mTOR activation was not sufficient to drive the malignant angiosarcoma phenotype. Genetic dissection of the spectrum of vascular tumors identified genes specifically regulated in the aggressive murine angiosarcomas that are also enriched in human angiosarcoma. The genetic dissection driving the transition across the malignant spectrum of endothelial sarcomas provides an opportunity to identify key determinants of the malignant phenotype, novel therapies for angiosarcoma, and novel in vivo models to further explore angiosarcoma pathogenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Role of proliferative marker index and KBTBD4 mutation in the pathological diagnosis of pineal parenchymal tumors.

Pineal parenchymal tumors (PPTs) are clinically rare and a biopsy is often required for a definitive diagnosis. To improve the accuracy of histological assessment of PPTs, we examined the proliferative capacity of PPT cells and investigated DICER1 expression and KBTBD4 mutations. This study included 19 cases of PPTs [3 pineocytomas (PCs), 10 PPTs of intermediate differentiation (PPTID), and 6 pineoblastomas (PBs)]. Immunohistochemistry for Ki-67, PHH3, and DICER1, as well as Sanger sequencing analysis for KBTBD4 mutations, was performed using formalin-fixed paraffin-embedded tissue specimens that were resected during surgery. Tumor cell proliferation was quantified using an image analysis software. For the PHH3 and MIB-1 indices, a significant difference was observed between the PPTIDs and PBs (P < 0.05). Loss of DICER1 was not specific for PB; 0/3 PCs (0.0%), 2/9 PPTIDs (22.2%), and 2/4 PBs (50.0%). KBTBD4 mutations were detected in 1/3 PCs (33.3%), 6/9 PPTIDs (66.7%), and 0/4 PBs (0.0%). Thus, combined application of the proliferative marker index and KBTBD4 mutation analysis may be useful for the differential diagnosis of PPTs. Furthermore, detection of KBTBD4 mutations using Sanger sequencing analysis may support the diagnosis of PPTID.

Hematologic indices in individuals with pathogenic germline DICER1 variants.

Pathogenic germline variants in DICER1 underlie an autosomal dominant, pleiotropic tumor-predisposition disorder. Murine models with the loss of DICER1 in hematopoietic stem cell progenitors demonstrate hematologic aberrations that include reductions in red and white blood cell counts, hemoglobin volume, and impaired maturation resulting in dysplasia. We investigated whether hematologic abnormalities such as those observed in DICER1-deficient mice were observed in humans with a pathogenic germline variant in DICER1. A natural history study of individuals with germline pathogenic DICER1 variants and family controls conducted through the National Cancer Institute (NCI) evaluated enrollees at the National Institutes of Health Clinical Center during a comprehensive clinical outpatient visit that included collecting routine clinical laboratory studies. These were compared against normative laboratory values and compared between the DICER1 carriers and controls. There were no statistical differences in routine clinical hematology laboratory studies observed in DICER1 carriers and family controls. A review of the medical history of DICER1 carriers showed that none of the individuals in the NCI cohort developed myelodysplastic syndrome or leukemia. Query of the International Pleuropulmonary Blastoma/DICER1 Registry revealed 1 DICER1 carrier who developed a secondary leukemia after treatment of pleuropulmonary blastoma. We found limited evidence that the hematologic abnormalities observed in murine DICER1 models developed in our cohort of DICER1 carriers. In addition, no cases of myelodysplastic syndrome were observed in either the NCI cohort or the International Pleuropulmonary Blastoma/DICER1 Registry; 1 case of presumed secondary leukemia was reported. Abnormalities in hematologic indices should not be solely attributed to DICER1. This trial was registered at www.clinicaltrials.gov as #NCT01247597.

Pineoblastoma is uniquely tolerant of mutually exclusive loss of DICER1, DROSHA or DGCR8.

Pineoblastoma is uniquely tolerant of mutually exclusive loss of DICER1, DROSHA or DGCR8.

Dicer1 facilitates liver regeneration in a manner dependent on the inhibitory effect of miR-21 on Pten and Rhob expression

AimsTamoxifen-induced liver-specific Dicer1 deletion (iDicer1−/−) in mature mice may provide clues demonstrating the genuine effects of acute loss of Dicer1 and miRNAs in the liver regeneration process. Main methodsIn this study, mice with tamoxifen-induced Dicer1 deletion through the Cre/LoxP system were constructed and then underwent classic 70% partial hepatectomy or CCl4-induced liver injury. To rescue the inhibitory effect of Dicer1 ablation on liver regeneration, miR-21 agomir was injected into the tail vein of iDicer1−/− mice. Key findingsUnlike constitutive embryonic deletion of Dicer1, tamoxifen-induced Dicer1 deletion did not result in severe liver injury or lesions, providing an ideal model for investigating acute loss of Dicer1 and miRNAs in liver regeneration. Dicer1 deletion led to impaired liver regeneration through the inhibitory effect of miR-21 on PTEN and Rhob expression. SignificanceIn our previous study, we found that embryonic loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation, while the role of Dicer1 in liver regeneration remains largely unknown. We clearly identified the promotion effect of Dicer1 on liver regeneration by increasing miR-21 expression, which inhibits the expression of two negative cell proliferation regulators, Pten and Rhob.

Conditional Dicer1 depletion using Chrnb4-Cre leads to cone cell death and impaired photopic vision

Irreversible photoreceptor cell death is a major cause of blindness in many retinal dystrophies. A better understanding of the molecular mechanisms underlying the progressive loss of photoreceptor cells remains therefore crucial. Abnormal expression of microRNAs (miRNAs) has been linked with the aetiology of a number of retinal dystrophies. However, their role during the degenerative process remains poorly understood. Loss of cone photoreceptors in the human macula has the greatest impact on sight as these cells provide high acuity vision. Using a Chrnb4-cre; Dicerflox/flox conditional knockout mouse (Dicer CKO) to delete Dicer1 from cone cells, we show that cone photoreceptor cells degenerate and die in the Dicer-deleted retina. Embryonic eye morphogenesis appeared normal in Dicer CKO mice. Cone photoreceptor abnormalities were apparent by 3 weeks of age, displaying either very short or absent outer segments. By 4 months 50% of cones were lost and cone function was impaired as assessed by electroretinography (ERG). RNAseq analysis of the Dicer CKO retina revealed altered expression of genes involved in the visual perception pathway. These data show that loss of Dicer1 leads to early-onset cone cell degeneration and suggest that Dicer1 is essential for cone photoreceptor survival and homeostasis.

DICER1 mutation and pituitary prolactinoma

SummaryA young woman carrying germline DICER1 mutation was discovered to have a pituitary microprolactinoma when she became amenorrhoic. The mutation was identified as a result of family screening following the early death of the patient’s daughter with ovarian cancer. The patient was in follow-up screening for thyroid disease, and investigations were initiated when she became amenorrhoic. MR scan revealed a 6 mm diameter pituitary microadenoma and raised prolactin. The prolactin was efficiently suppressed with low-dose cabergoline, and her menstrual cycles resumed. Dicer is an RNase enzyme, which is essential for processing small non-coding RNAs. These molecules play pleiotropic roles in regulating gene expression, by targeting mRNA sequences for degradation. DICER1 plays different roles depending on cell context, but is thought to be a functional tumour suppressor gene. Accordingly, germline mutation in one DICER1 allele is insufficient for oncogenesis, and a second hit on the other allele is required, as a result of postnatal somatic mutation. Loss of DICER1 is linked to multiple tumours, with prominent endocrine representation. Multinodular goitre is frequent, with increased risk of differentiated thyroid cancer. Rare, developmental pituitary tumours are reported, including pituitary blastoma, but not reports of functional pituitary adenomas. As DICER1 mutations are rare, case reports are the only means to identify new manifestations and to inform appropriate screening protocols.Learning points:DICER1 mutations lead to endocrine tumours.DICER1 is required for small non-coding RNA expression.DICER1 carriage and microprolactinoma are both rare, but here are reported in the same individual, suggesting association.Endocrine follow-up of patients carrying DICER1 mutations should consider pituitary disease.

Dicer1 dysfunction promotes stemness and aggression in endometrial carcinoma.

Endometrial carcinoma is one of the most common gynecological malignancies, but the molecular events involved in the development and progression of endometrial carcinoma remain unclear. Dicer1 and cancer stem cells play important roles in cell motility and survival. This study investigated the role of the let-7 family and Dicer1 in the stemness of endometrial carcinoma cells. We profiled Dicer1 expression in clinical samples and explored its relationship with stem cell-associated markers and clinical parameters. We showed that Dicer1 dysfunction leads to the enrichment of tumor stemness features and tumor aggression both in vitro and in vivo. We also identified the mechanism related to this potential tumor-predisposing phenotype: loss of Dicer1 induced abnormal expression of the let-7 family, which comprises well-known tumor suppressors, thus regulating stemness in endometrial carcinoma cells.

Dicer-1 regulates proliferative potential of Drosophila larval neural stem cells through bantam miRNA based down-regulation of the G1/S inhibitor Dacapo

The present work elucidates the role of miRNA in cell cycle regulation during brain development in Drosophila. Here we report that lineage specific depletion of dicer-1, a classically acknowledged miRNA biogenesis protein in neuroblasts leads to a reduction in their numbers and size in the third instar larval central brain. These brains also showed lower number of mitotically active cells and when homozygous mitotic clones were generated in an otherwise heterozygous dicer-1 mutant background via MARCM technique, they showed reduced number of progeny cells in individual clones, substantiating the adverse effect of the loss of dicer-1 on the proliferative potential of neuroblasts. bantam miRNA, which has been classically reported to be involved in tissue growth was found to express in neuroblasts and undergo reduced expression in Dicer-1 depleted background in the third instar larval brain. Reduction in the number and proliferative potential of neuroblasts in bantam mutant background implies a pivotal role played by bantam miRNA in maintenance of neuroblast number. Since, in both Dicer-1 and bantam depleted genetic backgrounds, Dacapo, an inhibitor of cyclin E-Cdk complex, was found to have elevated expression, we put forward a molecular mechanism involving bantam-Dacapo-Cyclin E/Cdk complex that regulates the G1-S phase transition of Drosophila neuroblasts.

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.

Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21–mediated tumor suppressor Pdcd4 repression

AbstractThe modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage-tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4.

Loss of Dicer1 impairs hepatocyte survival and leads to chronic inflammation and progenitor cell activation.

To investigate the continuous hepatic histopathological processes which occur in response to the loss of Dicer1. We generated a hepatocyte-selective Dicer1 knockout mouse and observed the gradual hepatic histopathological changes in the mutant liver. Immunohistochemistry and Western blotting were performed to detect Dicer1 expression. We performed hematoxylin and eosin staining, Periodic acid-Schiff staining, Oil Red O staining, and Masson's trichrome staining to detect histological changes in Dicer1-deficient livers. Ki67 immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and Western blotting were used to determine hepatocyte proliferation and apoptosis. Serum biochemistry, cytokine assays, and flow cytometric analysis were performed to quantity liver necrosis and inflammation. Fibrogenic markers were determined by Western blotting and qPCR. CK19, CD133, and OV6 immunofluorescence were used to observe liver progenitor cells. Immunofluorescence and qPCR were performed to reveal embryonic gene expression. We also performed histological staining and Western blotting to analyze hepatocellular carcinoma (HCC) development. Dicer1 inactivation resulted in significant architecture disorganization and metabolism disruption in the liver. Dicer1 disruption impaired hepatocyte survival and resulted in profound cell apoptosis and continuous necrosis. In contrast to previous reports, the mutant liver exhibited chronic inflammation and progressive fibrosis, and could not be repopulated by Dicer1-positive cells. In addition, extensive activation of hepatic progenitor cells was observed. Primary HCC was observed as early as 4 mo after birth. Hepatic loss of Dicer1 results in complex chronic pathological processes, including hepatocyte death, inflammatory infiltration, chronic fibrosis, compensatory proliferation, progenitor activation, and spontaneous hepatocarcinogenesis.

DICER1 is essential for survival of postmitotic rod photoreceptor cells in mice.

Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.

An Lmx1b-miR135a2 Regulatory Circuit Modulates Wnt1/Wnt Signaling and Determines the Size of the Midbrain Dopaminergic Progenitor Pool

MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.

MiR-130b is an EMT-related microRNA that targets DICER1 for aggression in endometrial cancer

Endometrial cancer (EC) is the most common gynecologic malignancy, but the molecular events involved in the development and progression of EC remain unclear. Certain microRNAs (miRNAs) and DICER1 play important roles in cell motility and survival. This study investigated the role of miR-130b and DICER1 in EC. We profiled miR-130b and DICER1 expression in clinical samples explored its relationship with clinical parameters. A luciferase reporter assay assessed the miR-130b targeting potential of DICER1. We show both in vitro and in vivo that miR-130b overexpression along with DICER1 dysfunction leads to tumor aggression and miRNA synthesis abnormalities that are related to cancer hallmarks through DICER1-miRNAs axis modulation. We also identify the mechanism related to this potential tumor predisposing phenotype: miR-130b and loss of DICER1 induced abnormal expression of EMT-related genes, which constitutes a loop regulation of the miR-130b-DICER1-EMT axis.

Deregulated Sex Chromosome Gene Expression with Male Germ Cell-Specific Loss of Dicer1

MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs that mediate post-transcriptional gene silencing by inhibiting mRNA translation and promoting mRNA decay. DICER1, an RNase III endonuclease encoded by Dicer1, is required for processing short 21–22 nucleotide miRNAs from longer double-stranded RNA precursors. Here, we investigate the loss of Dicer1 in mouse postnatal male germ cells to determine how disruptions in the miRNA biogenesis pathway may contribute to infertility. Reduced levels of Dicer1 transcripts and DICER1 were confirmed in germ cell knock-out (GCKO) testes by postnatal day 18 (P18). Compared to wild-type (WT) at 8 weeks, GCKO males had no change in body weight; yet showed significant reductions in testis mass and sperm number. Histology and fertility tests confirmed spermatogenic failure in GCKO males. Array analyses at P18 showed that in comparison to WT testes, 75% of miRNA genes and 37% of protein coding genes were differentially expressed in GCKO testes. Among these, 96% of miRNA genes were significantly down-regulated, while 4% miRNA genes were overexpressed. Interestingly, we observed preferential overexpression of genes encoded on the sex chromosomes in GCKO testes, including more than 80% of previously identified targets of meiotic sex chromosome inactivation (MSCI). Compared to WT, GCKO mice showed higher percentages of germ cells at early meiotic stages (leptotene and zygotene) but lower percentages at later stages (pachytene, diplotene and metaphase I) providing evidence that deletion of Dicer1 leads to disruptions in meiotic progression. Therefore, deleting Dicer1 in early postnatal germ cells resulted in deregulation of transcripts encoded by genes on the sex chromosomes, impaired meiotic progression and led to spermatogenic failure and infertility.

Synthetic lethality between Rb, p53 and Dicer or miR-17-92 in retinal progenitors suppresses retinoblastoma formation.

Synthetic lethality is a promising strategy for specific targeting of cancer cells that carry mutations that are absent in normal cells. This approach may help overcome the challenge associated with targeting dysfunctional tumour suppressors, such as p53 and Rb (refs 1, 2). Here we show that Dicer1 targeting prevents retinoblastoma formation in mice by synthetic lethality with combined inactivation of p53 and Rb. Although Dicer1 functions as a haploinsufficient tumour suppressor, its complete loss of function is selected against during tumorigenesis(3-5). We show that Dicer1 deficiency is tolerated in Rb-deficient retinal progenitor cells harbouring an intact p53 pathway, but not in the absence of p53. This synthetic lethality is mediated by the oncogenic miR-17-92 cluster because its deletion phenocopies Dicer1 loss in this context. miR-17-92 inactivation suppresses retinoblastoma formation in mice and co-silencing of miR-17/20a and p53 cooperatively decreases the viability of human retinoblastoma cells. These data provide an explanation for the selective pressure against loss of Dicer1 during tumorigenesis and a proof-of-concept that targeting miRNAs may potentially represent a general approach for synthetic lethal targeting of cancer cells that harbour specific cancer-inducing genotypes.

Inactivation of Dicer1 has a severe cumulative impact on the formation of mature germ cells in mouse testes

Dicer1, an RNase III endonuclease, is indispensable for the maturation of miRNA and siRNA, which control gene expression through the RNAi pathway. The diverse functions of miRNA involving multiple developmental processes have been elucidated, but the role of Dicer1 in spermatogenesis is just beginning to be revealed. Mice lacking Dicer1 were reported to be embryonic lethal at E7.5. In the present study, mice with a Dicer1 conditional allele were crossed with Vasa-cre transgenic mice to delete Dicer1 as early as the prospermatogonia stage (at E15). At P40, seminiferous tubules of Dicer1 deficient mice showed several aberrant phenotypes. A large number of apoptotic germ cells were detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, but several events in meiosis of spermatocytes appeared unaffected. The mutant mice were found to be sterile, likely due to the extensive decrease in number and morphological abnormalities of mature sperm in the epididymis, which, together with the numerous haploid cells in the testis, indicated a severely affected transition from round to functional elongated spermatozoa. Additionally, we found milder phenotypes when Dicer1 was inactivated in later stages of spermatogenesis in Stra8-cre and Pgk2-cre transgenic mice. In conclusion, our findings suggest that the loss of Dicer1 has a continuous and cumulative effect on the process of spermatogenesis and blocks the germ cells in the stage of round spermatids to a large extent, ultimately leading to the generation of abnormal sperm.

RNA interference in mammalian DNA methylation1This review is part of Special Issue entitled Asilomar Chromatin and has undergone the Journal’s usual peer review process.

RNAi and Dicer-dependent siRNAs are required for constitutive heterochromatin formation in fission yeast and for establishing DNA methylation at repetitive elements in plants. In the mammalian male germ line, DICER1-independent piRNAs are required for the full establishment of DNA methylation of dispersed repetitive transposable elements. However, in other mammalian cell types, no clear picture has yet emerged of the role of RNAi in establishing heterochromatin and DNA methylation. In mouse embryonic stem cells, which remain viable on loss of DICER1 and ablation of RNAi, while no firm evidence has been obtained for defective heterochromatin formation, there are indications of defective DNA methylation. The latter has been attributed to an indirect effect of reduced DNA methyltransferase (DNMT) activity due to a loss of miRNA-mediated gene regulation. However, it is unclear whether the reductions in DNMT activity were sufficient to affect DNA methylation. We consider it equally likely that the defects in DNA methylation that can be observed in DICER1-deficient embryonic stem cells are the result of nonspecific effects related to RNAi loss aside from reduced DNMT activity.