MiR-690, an exosomal-derived miRNA from M2-polarized macrophages, improves insulin sensitivity in obese mice.

Insulin resistance is a major pathophysiologic defect in type 2 diabetes and obesity, while anti-inflammatory M2-like macrophages are important in maintaining normal metabolic homeostasis. Here, we show that M2 polarized bone marrow-derived macrophages (BMDMs) secrete miRNA-containing exosomes (Exos), which improve glucose tolerance and insulin sensitivity when given to obese mice. Depletion of their miRNA cargo blocks the ability of M2 BMDM Exos to enhance insulin sensitivity. We found that miR-690 is highly expressed in M2 BMDM Exos and functions as an insulin sensitizer both in vivo and in vitro. Expressing an miR-690 mimic in miRNA-depleted BMDMs generates Exos that recapitulate the effects of M2 BMDM Exos on metabolic phenotypes. Nadk is a bona fide target mRNA of miR-690, and Nadk plays a role in modulating macrophage inflammation and insulin signaling. Taken together, these data suggest miR-690 could be a new therapeutic insulin-sensitizing agent for metabolic disease.

Tissue-specific and transcription-dependent mechanisms regulate primary microRNA processing efficiency of the human chromosome 19 MicroRNA cluster.

One of the longest human microRNA (miRNA) clusters is located on chromosome 19 (C19MC), containing 46 miRNA genes, which were considered to be expressed simultaneously and at similar levels from a common long noncoding transcript. Investigating the two tissue types where C19MC is exclusively expressed, we could show that there is a tissue-specific and chromosomal position-dependent decrease in mature miRNA levels towards the 3' end of the cluster in embryonic stem cells but not in placenta. Although C19MC transcription level is significantly lower in stem cells, this gradual decrease is not present at the primary miRNA levels, indicating that a difference in posttranscriptional processing could explain this observation. By depleting Drosha, the nuclease component of the Microprocessor complex, we could further enhance the positional decrease in stem cells, demonstrating that a tissue-specific, local availability of the Microprocessor complex could lie behind the phenomenon. Moreover, we could describe a tissue-specific promoter being exclusively active in placenta, and the epigenetic mark analysis suggested the presence of several putative enhancer sequences in this region. Performing specific chromatin immunoprecipitation followed by quantitative real-time PCR experiments we could show a strong association of Drosha with selected enhancer regions in placenta, but not in embryonic stem cells. These enhancers could provide explanation for a more efficient co-transcriptional recruitment of the Microprocessor, and therefore a more efficient processing of pri-miRNAs throughout the cluster in placenta. Our results point towards a new model where tissue-specific, posttranscriptional 'fine-tuning' can differentiate among miRNAs that are expressed simultaneously from a common precursor.

Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells.

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.

Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise.

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.

Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.

Deficiency of DICER reduces the invasion ability of trophoblasts and impairs the pro-angiogenic effect of trophoblast-derived microvesicles.

DICER is a key rate-limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER-dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh-Dicer) was partly due to the up-regulation of miR-16-2-3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh-Dicer HTR8 cells have an anti-angiogenic effect, due to reduced angiogenic factors and increased anti-angiogenic miRNAs (including let-7d, miR-1-6-2 and miR-15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER-miR-16-2-COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER-containing MVs mediate the pro-angiogenic effect of trophoblast-derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.

MicroRNA-10a-5p regulates macrophage polarization and promotes therapeutic adipose tissue remodeling.

OBJECTIVE: This study investigated the role of microRNAs generated from adipose tissue macrophages (ATMs) during adipose tissue remodeling induced by pharmacological and nutritional stimuli. METHODS: Macrophage-specific Dicer knockout (KO) mice were used to determine the roles of microRNA generated in macrophages in adipose tissue remodeling induced by the ß3-adrenergic receptor agonist CL316,243 (CL). RNA-seq was performed to characterize microRNA and mRNA expression profiles in isolated macrophages and PDGFRα+ adipocyte stem cells (ASCs). The role of miR-10a-5p was further investigated in cell culture, and in adipose tissue remodeling induced by CL treatment and high fat feeding. RESULTS: Macrophage-specific deletion of Dicer elevated pro-inflammatory gene expression and prevented CL-induced de novo beige adipogenesis in gonadal white adipose tissue (gWAT). Co-culture of ASCs with ATMs of wild type mice promoted brown adipocyte gene expression upon differentiation, but co-culture with ATMs of Dicer KO mice did not. Bioinformatic analysis of RNA expression profiles identified miR-10a-5p as a potential regulator of inflammation and differentiation in ATMs and ASCs, respectively. CL treatment increased levels of miR-10a-5p in ATMs and ASCs in gWAT. Interestingly, CL treatment elevated levels of pre-mir-10a in ATMs but not in ASCs, suggesting possible transfer from ATMs to ASCs. Elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured RAW 264.7 macrophages and promoted the differentiation of C3H10T1/2 cells into brown adipocytes. Furthermore, treatment with a miR-10a-5p mimic in vivo rescued CL-induced beige adipogenesis in Dicer KO mice. High fat feeding reduced miR-10a-5p levels in ATMs of gWAT, and treatment of mice with a miR-10a-5p mimic suppressed pro-inflammatory responses, promoted the appearance of new white adipocytes in gWAT, and improved systemic glucose tolerance. CONCLUSIONS: These results demonstrate an important role of macrophage-generated microRNAs in adipogenic niches and identify miR-10a-5p as a key regulator that reduces adipose tissue inflammation and promotes therapeutic adipogenesis.

A Direct RNA-to-RNA Replication System for Enhanced Gene Expression in Bacteria.

A long-standing objective of metabolic engineering has been to exogenously increase the expression of target genes. In this research, we proposed the permanent RNA replication system using DNA as a template to store genetic information in bacteria. We selected Qß phage as the RNA replication prototype and made many improvements to achieve target gene expression enhancement directly by increasing mRNA abundance. First, we identified the endogenous gene Rnc, the knockout of which significantly improved the RNA replication efficiency. Second, we elucidated the essential elements for RNA replication and optimized the system to make it more easily applicable. Combined with optimization of the host cell and the system itself, we developed a stable RNA-to-RNA replication tool to directly increase the abundance of the target mRNA and subsequently the target protein. Furthermore, it was proven efficient in enhancing the expression of specific proteins and was demonstrated to be applicable in metabolic engineering. Our system has the potential to be combined with any of the existing methods for increasing gene expression.

MiR-34 and MiR-200: Regulator of Cell Fate Plasticity and Neural Development.

Studies from last two decades have established microRNAs (miRNAs) as the most influential regulator of gene expression, especially at the post-transcriptional stage. The family of small RNA molecules including miRNAs is highly conserved and expressed throughout the multicellular organism. MiRNAs regulate gene expression by binding to 3' UTR of protein-coding mRNAs and initiating either decay or movement of mRNAs to stress granules. Tissues or cells, which go through cell fate transformation like stem cells, brain cells, iPSCs, or cancer cells show very dynamic expression profile of miRNAs. Inability to pass the developmental stages of Dicer (miRNA maturation enzyme) knockout animals has confirmed that expression of mature and functional miRNAs is essential for proper development of different organs and tissues. Studies from our laboratory and elsewhere have demonstrated the role of miR-200 and miR-34 families in neural development and have shown higher expression of both families in mature and differentiated neurons. In present review, we have provided a general overview of miRNAs and focused on the role of miR-34 and miR-200, two miRNA families, which have the capability to change the phenotype and fate of a cell in different tissues and situations.

MicroRNAs control eyelid development through regulating Wnt signaling.

BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

DICER1 regulates antibacterial function of epididymis by modulating transcription of ß-defensins.

DICER1 is a key enzyme responsible for the maturation of microRNAs. Recent evidences suggested that DICER1 and microRNAs expressed in epididymis were involved in the control of male fertility. However, the exact mechanism remains to be elucidated. Here, we created a mouse line by targeted disruption of Dicer1 gene in the principal cells of distal caput epididymis. Our data indicated that a set of ß-defensin genes were downregulated by DICER1 rather than by microRNAs. Moreover, DICER1 was significantly enriched in the promoter of ß-defensin gene and controlled transcription. Besides, the antibacterial ability of the adult epididymis significantly declined upon Dicer1 deletion both in vitro and in vivo. And a higher incidence of reproductive defect was observed in middle-aged Dicer1-/- males. These results suggest that DICER1 plays an important role in transcription of ß-defensin genes, which are associated with the natural antibacterial properties in a microRNA-independent manner, and further impacts the male fertility.

An inducible knockout of Dicer in adult mice does not affect endurance exercise-induced muscle adaptation.

The contractile and metabolic properties of adult skeletal muscle change in response to endurance exercise. The mechanisms of transcriptional regulation in exercise-induced skeletal muscle adaptation, including fiber-type switching and mitochondrial biogenesis, have been investigated intensively, whereas the role of microRNA (miRNA)-mediated posttranscriptional gene regulation is less well understood. We used tamoxifen-inducible Dicer1 knockout (iDicer KO) mice to reduce the global expression of miRNAs in adult skeletal muscle and subjected these mice to 2 wk of voluntary wheel running. Dicer mRNA expression was completely depleted in fast-twitch plantaris muscle after tamoxifen injection. However, several muscle-enriched miRNAs, including miR-1 and miR-133a, were reduced by only 30-50% in both the slow and fast muscles. The endurance exercise-induced changes that occurred for many parameters (i.e., fast-to-slow fiber-type switch and increases in succinate dehydrogenase, respiratory chain complex II, and citrate synthase activity) in wild type (WT) also occurred in the iDicer KO mice. Protein expression of myosin heavy chain IIa, peroxisome proliferator-activated receptor-γ coactivator-1α, and cytochrome c complex IV was also increased in the iDicer KO mice by the voluntary running. Furthermore, there was no significant difference in oxygen consumption rate in the isolated mitochondria between the WT and iDicer KO mice. These data indicate that muscle-enriched miRNAs were detectable even after 4 wk of tamoxifen treatment and there was no apparent specific endurance-exercise-induced muscle phenotype in the iDicer KO mice.

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.

Dicer promotes endothelial recovery and limits lesion formation after vascular injury through miR-126-5p.

BACKGROUND: The recovery of endothelial cells (ECs) after vascular injury is mainly mediated by the proliferation of resident ECs, thereby reducing neointima formation. The RNase Dicer processes microRNAs (miRNAs) and regulates EC function by controlling miRNA-mediated regulation of gene expression. This study aimed to investigate the impact of miRNA biogenesis in ECs on endothelial repair during lesion formation after vascular injury. METHODS AND RESULTS: To study the effect of Dicer on ECs during neointima formation, conditional deletion of Dicer was induced in Apoe-/- mice (EC-Dicerflox) by tamoxifen injection. Following wire-induced injury to carotid arteries of EC-Dicerflox mice, the EC recovery was impaired and the neointima formation and lesional macrophage accumulation was increased. Moreover, conditional deletion of Dicer in ECs diminished the expression of miR-126-5p in EC-Dicerflox mice. Notably, reconstitution of miR-126-5p in the injured arteries of EC-Dicerflox mice using miR-126-5p mimic, prevented the impaired endothelial recovery and increased lesion formation observed in EC-Dicerflox mice. CONCLUSIONS: Deficiency of endothelial Dicer diminished endothelial recovery and promoted neointima formation probably due to impaired miR-126-5p expression. Treatment with miR-126-5p mimics promotes endothelial recovery and thereby limits neointima formation. Thus, miR-126-5p therapy represents a potential approach to improve endothelial recovery and prevent restenosis following vascular injury.

Loss of Drosha underlies dopaminergic neuron toxicity in models of Parkinson's disease.

MiRNAs, a group of powerful modulator of gene expression, participate in multiple cellular processes under physiological and pathological conditions. Emerging evidence shows that Drosha, which controls the initial step in canonical miRNA biogenesis, is involved in modulating cell survival and death in models of several diseases. However, the role of Drosha in Parkinson's disease (PD) has not been well established. Here, we show that the level of Drosha decreases in 6-OHDA-induced cellular and animal models of PD. 6-OHDA induced a p38 MAPK-dependent phosphorylation of Drosha. This triggered Drosha degradation. Enhancing the level of Drosha protected the dopaminergic (DA) neurons from 6-OHDA-induced toxicity in both in vitro and in vivo models of PD and alleviated the motor deficits of PD mice. These findings reveal that Drosha plays a critical role in the survival of DA neurons and suggest that stress-induced destabilization of Drosha may be part of the pathological process in PD.

The deletion of dicer in mature myelinating glial cells causes progressive axonal degeneration but not overt demyelination in adult mice.

Myelinating glial cells (MGCs), oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS), generate myelin sheaths that insulate axons. After myelination is completed in adulthood, MGC functions independent from myelin are required to support axon survival, but the underlying mechanisms are still unclear. Dicer is a key enzyme that is responsible for generating functional micro-RNAs (miRNAs). Despite the importance of Dicer in initiating myelination, the role of Dicer in mature MGCs is still unclear. Here, Dicer was specifically deleted in mature MGCs in 2-month old mice (PLP-CreERT; Dicer fl/fl) by tamoxifen administration. Progressive motor dysfunction was observed in the Dicer conditional knockout mice, which displayed hind limb ataxia at 3 months post recombination that deteriorated into paralysis within 5 months. Massive axonal degeneration/atrophy in peripheral nerves was responsible for this phenomenon, but overt demyelination was not observed in either the CNS or PNS. In contrast to the PNS, signs of axonal degeneration were not observed in the CNS of these animals. We induced a Dicer deletion in oligodendroglia at postnatal day 5 in NG2-CreERT; Dicer fl/fl mice to evaluate whether Dicer expression in OLs is essential for axonal survival. Dicer deletion in oligodendroglia did not cause motor dysfunction at the age of 7 months. Neither axonal atrophy nor demyelination was observed in the CNS. Based on our results, Dicer expression in SCs is required to maintain axon integrity in adult PNS, and Dicer is dispensable for maintaining myelin sheaths in MGCs.

Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum.

This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, dicer genes (dcl-1, dcl-2, and both dcl-1/dcl-2) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2. Complementation of a single dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.

Egr2-dependent microRNA-138 is dispensable for peripheral nerve myelination.

Recent studies have elucidated the crucial role for microRNAs in peripheral nerve myelination by ablating components of the microRNA synthesis machinery. Few studies have focused on the role of individual microRNAs. To fill this gap, we focused this study on miR-138, which was shown to be drastically reduced in Dicer1 and Dgcr8 knockout mice with hypomyelinating phenotypes and to potentially target the negative regulators of Schwann cell differentiation. Here, we show that of two miR-138 encoding loci, mir-138-1 is the predominant locus transcribed in Schwann cells. mir-138-1 is transcriptionally upregulated during myelination and downregulated upon nerve injury. EGR2 is required for mir-138-1 transcription during development, and both SOX10 and EGR2 bind to an active enhancer near the mir-138-1 locus. Based on expression analyses, we hypothesized that miR-138 facilitates the transition between undifferentiated Schwann cells and myelinating Schwann cells. However, in conditional knockouts, we could not detect significant changes in Schwann cell proliferation, cell cycle exit, or myelination. Overall, our results demonstrate that miR-138 is an Egr2-dependent microRNA but is dispensable for Schwann cell myelination.

RNA Interference-Mediated Gene Silencing by Branched Tripodal RNAs Does Not Require Dicer Processing.

Specific gene silencing through RNA interference (RNAi) holds great promise as the next-generation therapeutic development platform. Previously, we have shown that branched, tripodal interfering RNA (tiRNA) structures could simultaneously trigger RNAi-mediated gene silencing of three target genes with 38 nt-long guide strands associated with Argonaute 2. Herein, we show that the branched RNA structure can trigger effective gene silencing in Dicer knockout cell line, demonstrating that the Dicer-mediated processing is not required for tiRNA activity. The finding of this study confirms the flexibility of the structure of RNAi triggers as well as the length of the guide strand in RNAi-mediated gene silencing.

Dicer1 ablation in osterix positive bone forming cells affects cortical bone homeostasis.

The RNAse III enzyme Dicer plays a major role in the processing of microRNAs from large pre-miRNAs. Dicer1 processed microRNAs are known to play a comprehensive role in osteoblast differentiation, bone remodeling and skeletal disorders. Targeted deletion of Dicer1 in osteo-progenitor cells is deleterious to fetal survival whereas targeted deletion in mature osteoblasts leads to an increase in bone mass. To address the role of Dicer1 in post-natal skeletal homeostasis, we generated a pre-osteoblast specific Dicer1 knockout model employing Tamoxifen controllable Cre allele, enabling us, via tamoxifen administration, to time-controllably ablate Dicer1 gene expression in osterix expressing bone forming cells in post-natal mice. Inactivation of Dicer1 in osterix positive bone forming cells led to striking dysregulation of cortical bone formation in pre-pubertal as well as adult mice. Cortical bone thickness was found to be significantly decreased in the Cre+ femora of both young and adult mice. Further, biomechanical testing experiments showed increased ductility, reduced stiffness and altered load at upper yield among the Cre+ tibiae. Our results suggest that Dicer1 processed microRNAs might play an important role in the regulation of post-natal cortical bone formation.