Dicer Deficiency Research Articles

Global Switch from DICER-dependent MicroRNA to DICER-independent SnoRNA-derived RNA Biogenesis in Malignancy.

SnoRNAs are frequently processed into snoRNA-derived RNAs (sdRNAs) that function much like traditional microRNAs (miRNAs). That said, our analyses suggest a global switch from DICER-dependent (predominately miRNA) to DICER-independent (predominately sdRNA) biogenesis/gene regulation in colon cancer. Whereas the expressions of 259 of 288 appreciably expressed miRNAs are significantly decreased (avg. 6.4% of WT) in human colon cancer DICER-KOs, 95 of 103 sdRNAs are conversely, significantly increased (avg. 679.3%) in DICER-KOs as compared to WT. As many diseases are characterized by DICER deficiency, this putative global switch to DICER-independent sdRNA regulations may contribute to an array of human diseases.

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.

Significant Role of Dicer and miR-223 in Adipose Tissue of Polycystic Ovary Syndrome Patients.

Polycystic ovary syndrome (PCOS) is a chronic metabolic disease that is associated with obesity and adipose tissue dysfunction. This study aimed to explore the roles of Dicer (an enzyme that processes primary microRNAs) and microRNAs in PCOS. Protein levels were detected by western blotting, and mRNA and microRNA levels were detected by RT-PCR. Dicer-deficient pre-adipocytes were established by lentiviral transfection, and an miR-223 mimic and miR-223 inhibitor were used to overexpress and inhibit miR-223, respectively. 3T3-L1 cells were induced to differentiate into mature adipocytes by IBMX, insulin, and dexamethasone. The degree of differentiation was determined by oil red O staining. An insulin resistance model was established by exposing mature adipocytes to excessive glucose and insulin. The protein levels of Dicer and Ago2 in adipose tissues of PCOS patients were significantly lower than those in control females. A Dicer-deficient 3T3-L1 cell model was successfully established, whose proliferation was inhibited significantly. Insulin-resistant mature adipocytes expressed significantly less Dicer protein than control cells. The differentiation of Dicer-deficient 3T3-L1 cells and their expression of miR-223 and marker genes associated with adipose differentiation were reduced significantly. Furthermore, 3T3-L1 cells showed a weaker ability to develop into mature adipocytes when miR-223 expression was inhibited. An miR-223 mimic was used to recover the differentiation block induced by Dicer deficiency. This rescued the expression of genes associated with adipose differentiation, although the differentiation block was not efficiently rescued. It is concluded that insulin resistance may contribute to the decreased levels of Dicer protein in adipose tissue of PCOS patients. This suggests that dysfunction of Dicer plays a significant role in obesity of PCOS patients. miR-223 is a key factor in Dicer-regulated adipose differentiation, and other microRNAs may be involved in the process.

Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3.

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

Mesenchymal cell differentiation and diseases: involvement of translin/TRAX complexes and associated proteins.

Translin and translin-associated factor X (translin/TRAX) proteins have been implicated in a variety of cellular activities central to nucleic acid metabolism. Accumulating evidence indicates that translin/TRAX complexes participate in processes ensuring the replication of DNA, as well as cell division. Significant progress has been made in understanding the roles of translin/TRAX complexes in RNA metabolism, such as through RNA-induced silencing complex activation or the microRNA depletion that occurs in Dicer deficiency. At the cellular level, translin-deficient (Tsn-/- ) mice display delayed endochondral ossification or progressive bone marrow failure with ectopic osteogenesis and adipogenesis, suggesting involvement in mesenchymal cell differentiation. In this review, we summarize the molecular and cellular functions of translin homo-octamer and translin/TRAX hetero-octamer. Finally, we discuss the multifaceted roles of translin, TRAX, and associated proteins in the healthy and disease states.

Dicer Deficiency Differentially Impacts Microglia of the Developing and Adult Brain

Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.

Calvarial bone development and suture closure in Dicer-deficient mice.

To evaluate whether lack of Dicer during calvaria development would lead to dysmorphology of calvaria and suture closure in mice. A conditional Dicer deficient under Osx promoter mouse was employed in this study. The 4- and 10-week-old conditional Dicer-deficient mice control littermates and Osx-cre transgenic mice were studied for calvarial bone morphology and suture closure. Dry skull, microcomputed tomography (μCT), histological and gene expression studies were investigated to evaluate the effect of Dicer deficiency on calvarial bone morphology and their related genes during calvaria development. The results elucidated that complete suture closure was observed in 10-week-old conditional Dicer-deficient mice while incomplete closure suture was observed in age-matched Osx-cre control mice. The μCT and histological sections demonstrated complete fusion of posterior frontal suture and dysmorphic calvarial bones in Dicer deficient mice compared to the ones in their littermates and age-matched Osx-cre control mice. Gene expression study demonstrated significantly increased expression of suture and calvarial bone-related genes, that is Tgf-beta family, Bmp3, Msx2, Alx4, Runx2 and Osx in Dicer-deficient mice during suture closure time. The results suggest mature miRNAs are important for suture closure and calvarial morphology during calvaria development.

Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals

RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.

Suppression of microRNA activity amplifies IFN-γ-induced macrophage activation and promotes anti-tumour immunity.

Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeuticimplications.

Fat-specific Dicer deficiency accelerates aging and mitigates several effects of dietary restriction in mice.

Aging increases the risk of type 2 diabetes, and this can be prevented by dietary restriction (DR). We have previously shown that DR inhibits the downregulation of miRNAs and their processing enzymes - mainly Dicer - that occurs with aging in mouse white adipose tissue (WAT). Here we used fat-specific Dicer knockout mice (AdicerKO) to understand the contributions of adipose tissue Dicer to the metabolic effects of aging and DR. Metabolomic data uncovered a clear distinction between the serum metabolite profiles of Lox control and AdicerKO mice, with a notable elevation of branched-chain amino acids (BCAA) in AdicerKO. These profiles were associated with reduced oxidative metabolism and increased lactate in WAT of AdicerKO mice and were accompanied by structural and functional changes in mitochondria, particularly under DR. AdicerKO mice displayed increased mTORC1 activation in WAT and skeletal muscle, where Dicer expression is not affected. This was accompanied by accelerated age-associated insulin resistance and premature mortality. Moreover, DR-induced insulin sensitivity was abrogated in AdicerKO mice. This was reverted by rapamycin injection, demonstrating that insulin resistance in AdicerKO mice is caused by mTORC1 hyperactivation. Our study evidences a DR-modulated role for WAT Dicer in controlling metabolism and insulin resistance.

Endothelial Dicer promotes atherosclerosis and vascular inflammation by miRNA-103-mediated suppression of KLF4.

MicroRNAs regulate the maladaptation of endothelial cells (ECs) to naturally occurring disturbed blood flow at arterial bifurcations resulting in arterial inflammation and atherosclerosis in response to hyperlipidemic stress. Here, we show that reduced endothelial expression of the RNAse Dicer, which generates almost all mature miRNAs, decreases monocyte adhesion, endothelial C–X–C motif chemokine 1 (CXCL1) expression, atherosclerosis and the lesional macrophage content in apolipoprotein E knockout mice (Apoe−/−) after exposure to a high-fat diet. Endothelial Dicer deficiency reduces the expression of unstable miRNAs, such as miR-103, and promotes Krüppel-like factor 4 (KLF4)-dependent gene expression in murine atherosclerotic arteries. MiR-103 mediated suppression of KLF4 increases monocyte adhesion to ECs by enhancing nuclear factor-κB-dependent CXCL1 expression. Inhibiting the interaction between miR-103 and KLF4 reduces atherosclerosis, lesional macrophage accumulation and endothelial CXCL1 expression. Overall, our study suggests that Dicer promotes endothelial maladaptation and atherosclerosis in part by miR-103-mediated suppression of KLF4.

Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum.

Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

Drosha controls dendritic cell development by cleaving messenger RNAs encoding inhibitors of myelopoiesis.

To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.

Rescuing dicer Defects via Inhibition of an Anti-Dicing Nuclease

Genetic defects in the microRNA (miRNA) generating enzyme, dicer, are increasingly linked to disease. Loss of miRNA in dicer deficiency is thought to be due to loss of miRNA-generating activity. Here, we demonstrate a catabolic mechanism driving miRNA depletion in dicer deficiency. We developed a Dicer-antagonist assay revealing a pre-miRNA degrading enzyme that competes with pre-miRNA processing. We purified this pre-miRNA degrading activity using an unbiased chromatographic procedure and identified the ribonuclease complex Translin/Trax (TN/TX). In wild-type dicer backgrounds, pre-miRNA processing was dominant. However, in dicer-deficient contexts, TN/TX broadly suppressed miRNA. These findings indicate that miRNA depletion in dicer deficiency is due to the combined loss of miRNA-generating activity and catabolic function of TN/TX. Importantly, inhibition of TN/TX mitigated loss of both miRNA and tumor suppression with dicer haploinsufficiency. These studies reveal a potentially druggable target for restoring miRNA function in cancers and emerging dicer deficiencies.

Dicer Deficiency Reveals MicroRNAs Predicted to Control Gene Expression in the Developing Adrenal Cortex

MicroRNAs (miRNAs) are small, endogenous, non-protein-coding RNAs that are an important means of posttranscriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer-knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer-KO adrenals demonstrated a significant loss of steroidogenic factor 1-expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by staining of proliferating cell nuclear antigen. To further characterize the embryonic adrenals from Dicer-KO mice, we performed microarray analyses for both gene and miRNA expression on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer-KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21, that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer-KO adrenals. Together these data suggest a role for miRNA-mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis.

Drosha regulates neurogenesis by controlling Neurogenin 2 expression independent of microRNAs

Temporal regulation of embryonic neurogenesis is controlled by hypostable transcription factors. The mechanism of the process is unclear. Here we show that the RNase III Drosha and DGCR8 (also known as Pasha), key components of the microRNA (miRNA) microprocessor, have important functions in mouse neurogenesis. Loss of microprocessor in forebrain neural progenitors resulted in a loss of stem cell character and precocious differentiation whereas Dicer deficiency did not. Drosha negatively regulated expression of the transcription factors Neurogenin 2 (Ngn2) and NeuroD1 whereas forced Ngn2 expression phenocopied the loss of Drosha. Neurog2 mRNA contains evolutionarily conserved hairpins with similarities to pri-miRNAs, and associates with the microprocessor in neural progenitors. We uncovered a Drosha-dependent destabilization of Neurog2 mRNAs consistent with microprocessor cleavage at hairpins. Our findings implicate direct and miRNA-independent destabilization of proneural mRNAs by the microprocessor, which facilitates neural stem cell (NSC) maintenance by blocking accumulation of differentiation and determination factors.

Re-dicing the pancreatic β-cell: do microRNAs define cellular identity?

Re-dicing the pancreatic β-cell: do microRNAs define cellular identity?

Canonical and alternate functions of the microRNA biogenesis machinery

The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K-Cre knock-in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR-155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency-induced osteoclastic suppression was dominant over Dicer deficiency-induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen-Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo.