The Multifaceted Profile of Thyroid Disease in the Background of DICER1 Germline and Somatic Mutations: Then, Now and Future Perspectives

DICER1 protein is a member of the ribonuclease (RNAse) III family with a key role in the biogenesis of microRNAs (miRNA) and in microRNA processing, potentially affecting gene regulation at the post-transcriptional level. The role of DICER1 and its relevance to thyroid cellular processes and tumorigenesis have only recently been explored, following the acknowledgement that DICER1 germline and somatic changes can contribute not only to non-toxic multinodule goiter (MNG) lesions detected in individuals of affected families but also to a series of childhood tumours, including thyroid neoplasms, which can be identified from early infancy up until the decade of 40s. In a context of DICER1 germline gene mutation, thyroid lesions have recently been given importance, and they may represent either an index event within a syndromic context or the isolated event that may trigger a deeper and broader genomic analysis screening of individuals and their relatives, thereby preventing the consequences of a late diagnosis of malignancy. Within the syndromic context MNG is typically the most observed lesion. On the other hand, in a DICER1 somatic mutation context, malignant tumours are more common. In this review we describe the role of DICER protein, the genomic events that affect the DICER1 gene and their link to tumorigenesis as well as the frequency and pattern of benign and malignant thyroid lesions and the regulation of DICER1 within the thyroidal environment.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses—including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

High Glucose Treatment Limits Drosha Protein Expression and Alters AngiomiR Maturation in Microvascular Primary Endothelial Cells via an Mdm2-dependent Mechanism

Diabetes promotes an angiostatic phenotype in the microvascular endothelium of skeletal muscle and skin. Angiogenesis-related microRNAs (angiomiRs) regulate angiogenesis through the translational repression of pro- and anti-angiogenic genes. The maturation of micro-RNA (miRs), including angiomiRs, requires the action of DROSHA and DICER proteins. While hyperglycemia modifies the expression of angiomiRs, it is unknown whether high glucose conditions alter the maturation process of angiomiRs in dermal and skeletal muscle microvascular endothelial cells (MECs). Compared to 5 mM of glucose, high glucose condition (30 mM, 6–24 h) decreased DROSHA protein expression, without changing DROSHA mRNA, DICER mRNA, or DICER protein in primary dermal MECs. Despite DROSHA decreasing, high glucose enhanced the maturation and expression of one angiomiR, miR-15a, and downregulated an miR-15a target: Vascular Endothelial Growth Factor-A (VEGF-A). The high glucose condition increased Murine Double Minute-2 (MDM2) expression and MDM2-binding to DROSHA. Inhibition of MDM2 prevented the effects evoked by high glucose on DROSHA protein and miR-15a maturation in dermal MECs. In db/db mice, blood glucose was negatively correlated with the expression of skeletal muscle DROSHA protein, and high glucose decreased DROSHA protein in skeletal muscle MECs. Altogether, our results suggest that high glucose reduces DROSHA protein and enhances the maturation of the angiostatic miR-15a through a mechanism that requires MDM2 activity.

EPCO-39. DICER1 SYNDROME WITH PRIMITIVE NEUROECTODERMAL TUMOR FROM SCREENING MUTATIONS OF DICER1 GENE

BACKGROUND DICER1 syndrome is a rare disorder of tumor predisposition. The DICER1 gene encodes the Dicer protein of the ribonuclease Ⅲ family and plays a critical role in miRNA creation. Patients with DICER1 syndrome-related cancer commonly harbor an additional somatic variant in exon24 or 25 of DICER1 involving one of the hotspots including p.E1705, p.D1709, p.G1809, p.D1810, or p.E1813. Herein, we firstly report the presence of hotspot missence mutation in a patient with primitive neuroectodermal tumor (PNET). METHODS The mutations in DIRCER1 were searched in the tumor tissue or peripheral blood from 4,349 patients with different types of cancer via next generation sequencing. Among them, comprehensive genomic profiling (CGP) with 131 CNS cancer-related genes and 4 brain tumor-related chromosome structure variations was performed in 230 cases, and CGP with 539 cancer-related genes was administrated in 4,119 cases. The histopathology diagnosis of each case was confirmed via hematoxylin and eosin staining. RESULTS A number of 99 mutations in the exon and intron regions of DICER1 gene were observed from 88 patients in 4,349 cases. The ratio of somatic variants located on exon 24 and 25 where both encoded the RNase Ⅲb cleavage domain to all mutations was 9.09% (9/99). There were 3 and 6 somatic variants in the exon 24 and 25, respectively. Among the 9 mutations, 4 of them were p.E1813, one class of hotspots located on exon 25 leading to DICER1 syndrome, and the other 4 hotspots were not observed. Among 4 cases of DICER1 syndrom, pleuropulmenary blastoma was common, rhabdomyosarcomas arisen in two cases were also reported before, and the last one was patient with PNET. CONCLUSIONS In this study, we analyze the mutation distribution of DICER1 in cancer patients, report firstly a patient with PNET harbors a hotspot, and widen and deepen the understanding of DICER1 syndrome.

Dicing the Disease with Dicer: The Implications of Dicer Ribonuclease in Human Pathologies

Gene expression dictates fundamental cellular processes and its de-regulation leads to pathological conditions. A key contributor to the fine-tuning of gene expression is Dicer, an RNA-binding protein (RBPs) that forms complexes and affects transcription by acting at the post-transcriptional level via the targeting of mRNAs by Dicer-produced small non-coding RNAs. This review aims to present the contribution of Dicer protein in a wide spectrum of human pathological conditions, including cancer, neurological, autoimmune, reproductive and cardiovascular diseases, as well as viral infections. Germline mutations of Dicer have been linked to Dicer1 syndrome, a rare genetic disorder that predisposes to the development of both benign and malignant tumors, but the exact correlation of Dicer protein expression within the different cancer types is unclear, and there are contradictions in the data. Downregulation of Dicer is related to Geographic atrophy (GA), a severe eye-disease that is a leading cause of blindness in industrialized countries, as well as to psychiatric and neurological diseases such as depression and Parkinson’s disease, respectively. Both loss and upregulation of Dicer protein expression is implicated in severe autoimmune disorders, including psoriasis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis and autoimmune thyroid diseases. Loss of Dicer contributes to cardiovascular diseases and causes defective germ cell differentiation and reproductive system abnormalities in both sexes. Dicer can also act as a strong antiviral with a crucial role in RNA-based antiviral immunity. In conclusion, Dicer is an essential enzyme for the maintenance of physiology due to its pivotal role in several cellular processes, and its loss or aberrant expression contributes to the development of severe human diseases. Further exploitation is required for the development of novel, more effective Dicer-based diagnostic and therapeutic strategies, with the goal of new clinical benefits and better quality of life for patients.

Silencing Dicer-Like Genes Reduces Virulence and sRNA Generation in Penicillium italicum, the Cause of Citrus Blue Mold

The Dicer protein is one of the most important components of RNAi machinery because it regulates the production of small RNAs (sRNAs) in eukaryotes. Here, Dicer1-like gene (Pit-DCL1) and Dicer2-like gene (Pit-DCL2) RNAi transformants were generated via pSilent-1 in Penicillium italicum (Pit), which is the causal agent of citrus blue mold. Neither transformant showed a change in mycelial growth or sporulation ability, but the pathogenicity of the Pit-DCL2 RNAi transformant to citrus fruits was severely impaired, compared to that of the Pit-DCL1 RNAi transformant and the wild type. We further developed a citrus wound-mediated RNAi approach with a double-stranded fragment of Pit-DCL2 generated in vitro, which achieved an efficiency in reducing Pi-Dcl2 expression and virulence that was similar to that of protoplast-mediated RNAi in P. italicum, suggesting that this approach is promising in the exogenous application of dsRNA to control pathogens on the surface of citrus fruits. In addition, sRNA sequencing revealed a total of 69.88 million potential sRNAs and 12 novel microRNA-like small RNAs (milRNAs), four of which have been predicated on target innate immunity or biotic stress-related genes in Valencia orange. These data suggest that both the Pit-DCL1 and Pit-DCL2 RNAi transformants severely disrupted the biogenesis of the potential milRNAs, which was further confirmed for some milRNAs by qRT-PCR or Northern blot analysis. These data suggest the sRNAs in P. italicum that may be involved in a molecular virulence mechanism termed cross-kingdom RNAi (ck-RNAi) by trafficking sRNA from P. italicum to citrus fruits.

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.

PTX3 Regulates miR-21 Expression and Secretion in Brown Adipocytes During Lipopolysaccharide-induced Inflammation (P21-072-19)

Objectives Metabolic endotoxemia is known to induce systemic inflammation and adipose tissue dysfunction during obesity. Pentraxin 3 (PTX3), a member of the pentraxin family can be induced by inflammatory stimuli in adipocytes. However, how PTX3 regulates inflammation in adipocytes is not fully understood. MicroRNAs (miRNAs) are key regulators of inflammatory gene expression. Specifically, miR-21 plays a role in the resolution of inflammation through inhibiting Toll-like receptor 4 (TLR4) signaling pathway. This study aimed to investigate how PTX3 regulates inflammatory homeostasis involving miR-21 during lipopolysaccharide (LPS) stimulation in brown adipocytes. Methods Using PTX3 knockout (KO) mouse and primary stromal-vascular (SV) cell culture models, we determined the effect of PTX3 deficiency on miR-21 expression, secretion, and inflammatory response in brown adipocytes, as well as the rescue effect of recombinant PTX3 on LPS-induced inflammation. Results Brown adipose tissue (BAT) had significantly higher levels of Dicer protein expression than inguinal and epididymal adipose depots; fully differentiated brown adipocytes expressed higher levels of Dicer than SV cells. These results suggest that BAT is the major site of miRNA production, and brown adipocyte is the main cell type that produces miRNAs. Moreover, we found brown adipocytes but not SV cells are the LPS responsive cells in miR-21 expression and secretion. In WT brown adipocytes, LPS stimulation significantly up-regulated cellular levels of miR-21. Interestingly, PTX3 deficiency led to a significant increase in the basal levels of both cellular and secreted miR21 compared to WT cells. Unlike in WT cells, LPS treatment suppressed cellular expression as well as secretion of miR-21 in PTX3 KO brown adipocytes. Treatment of recombinant PTX3 slightly up-regulated cellular expression of miR-21, but significantly increased the secretion of miR-21 in PTX3 KO brown adipocytes. Moreover, Treatment of recombinant PTX3 significantly attenuated LPS-stimulated increase in the expression of TNFα and MCP1 genes in PTX3 KO adipocytes. Conclusions We conclude that PTX3 plays an anti-inflammatory role in part via regulating the expression and secretion of miR-21. Funding Sources American Diabetes Association.