DICER1: The Argonaute Endonuclease Family Member and Its Role in Pediatric and Youth Pathology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Sergi and Minervini focused on the argonaute endonuclease family member and its role in pediatric and youth pathology. The Authors revealed the full range of DICER1 functions and the corresponding relationship to tumorigenic and non-neo plastic diseases. They postulated that the ubiquity of this enzyme in the human body makes it an exquisite target for nanotechnology supported therapies and repurposing drug approaches

From my point of view, the manuscript is interesting and will attract the interest of many scientists working with such systems. However, the manuscript contains sections that appear to be unoriginal, as indicated by the plagiarism report, having appeared in previously published works. For this reason, manuscript cannot be considered for publication in this form.  Please modify critical parts to reduce the overlap with the literature.  I recommend the manuscript for publication after major revision.

Comments on the Quality of English Language

Moderate editing of English language and style is required.

Author Response

In this manuscript, Sergi and Minervini focused on the argonauteendonuclease family member and its role in pediatric and youthpathology.
The Authors revealed the full range of DICER1 functionsand the corresponding relationship to tumorigenic and non-neoplastic diseases. They postulated that the ubiquity of this enzyme inthe human body makes it an exquisite target for nanotechnologysupported therapies and repurposing drug approaches
From my point of view, the manuscript is interesting and will attractthe interest of many scientists working with suchsystems.
However, the manuscript contains sections that appear tobe unoriginal, as indicated by the plagiarism report, havingappeared in previously published works.
For this reason,manuscript cannot be considered for publication in thisform.
Please modify critical parts to reduce the overlap with theliterature.
I recommend the manuscript for publication after majorrevision.

Thank you very much for your comments and suggestions. We revised the manuscript thoroughly to avoid plagiarism. From line number 25 through 465 the text was revised accordingly to reduce the overlap with the literature. We added more sections as requested by other reviewers.

Abstract: In 2001, two enzyme-encoding genes were recognized in the fruit fly Drosophila melanogaster. The genetic material, labeled Dicer-1 and Dicer-2, encodes ribonuclease-type enzymes with slightly diverse target substrates. The human orthologue is DICER1. It is a gene, which has been positioned on chromosome 14q32.13. It contains 27 exons, which are linking the two enzyme domains. DICER1 is found in all organ systems. It has been proved that it is paramount in human development. The protein determined by DICER1 is a ribonuclease (RNase). This RNase belongs to the RNase III superfamily, formally known as 'endoribonuclease'. It has been determined that the function of RNase III proteins is set to identify and degrade double-stranded molecules of RNA. DICER1 is a vital “housekeeping” gene. The multi-domain enzyme is key for small RNA processing. This enzyme functions in numerous pathways, including RNA interference paths, DNA damage renovation, and response to viruses. At the protein level, DICER is also involved in several human diseases, of which the pleuro-pulmonary blastoma is probably the most egregious entity. Numerous studies have determined the full range of DICER1 functions and the corresponding relationship to tumorigenic and non-neoplastic diseases. In fact, genetic mutations (somatic and germline) have been detected in DICER1 and are genetically associated with at least two clinical syndromes: DICER1 syndrome and GLOW syndrome. The ubiquity of this enzyme in the human body makes it an exquisite target for nanotechnology-supported therapies and repurposing drug approaches

Keywords: DICER1; oncogene; biology; molecular genetics

 

 

INTRODUCTION

Despite significant interest and research, DICER1 is a relatively new and developing factor in human disease. This review gives a thorough overview of the gene, encompassing its protein structure, associations, and involvement in disease. The prospect of targeting DICER1 and endoribonuclease DICER as therapeutic interventions is highly appealing. Through additional research, we may effectively utilize their widespread presence to treat many diseases. The DICER1 gene encodes a crucial protein in various biological processes. It is involved in producing small RNA molecules, which are essential for regulating gene expression and controlling cellular functions. Two genes encoding enzymes were discovered in D. melanogaster in 2001 [1]. The genes Dicer-1 and Dicer-2 produce ribonuclease enzymes. They target quite different substrates. The human orthologue is DICER1. It is a gene found on chromosome 14q32.13. DICER1 consists of 27 exons. It possesses two domains [2]. This gene exhibits expression in tissues derived from several organ systems and proper human development relies on this key gene. The protein encoded by DICER1 is an endoribonuclease. It belongs to the RNase III superfamily, also known as DICER. This set of proteins is found ubiquitously in eukaryotes. Prokaryotes lack an analogous protein, but they possess proteins that operate in comparable paths [3]. In general, the primary role of RNase III proteins is to identify and split apart double-stranded molecules of RNA [3].

In humans, the DICER molecule has an "L" shape, consisting of a head, body, and base. The substrate, often dsRNA (double strand RNA) or pre-miRNA, is located in the main body of the molecule (Figure 1).

 

Figure 1. RNAi. RNA interference (RNAi), also known as PTGS or Post-Transcriptional Gene Silencing (PTGS) or Genetic Perturbation Platform, is a preserved biological mechanism that responds to dsRNA or double-stranded RNA, enabling resistance to both parasite of endogenous origin and exogenous harmful nucleic acids, while regulating the expression of genes, which codify proteins. This innate process for sequence-explicit gene silencing has the potential to transform experimental biology. It  may have significant applications in genomics and functional genomics, as well as therapeutic intervention and other fields. Endogenous activators of the RNAi path including foreign DNA or dsRNA of viral origin, inconsistent transcripts from repeating genomic sequences (e.g., transposons), and pre-miRNA (miRNA, microRNA). In plants, RNAi underpins virus-induced gene silencing (VIGS), indicating a significant role in disease resistance. Investigations on C. elegans has proposed a potential method for the control of endogenous genes by the RNAi mhaveachinery. In mammalian cells, large double-stranded RNAs (>30 nt) typically elicit an interferon response. A streamlined model for the RNAi process consists of two stages. Both stages involve a ribonuclease enzyme. The first step is for the RNase II enzymes Dicer and Drosha to transform the trigger RNA (which could be dsRNA or miRNA primary transcript) into short interference RNA (siRNA). The RNA-induced silencing complex (RISC) is an effector complex that incorporates siRNAs in the following step. As the RISC is assembled, the siRNA is unwound, allowing the single-stranded RNA to hybridize with the mRNA target. Argonaute, an RNase H enzyme, nucleolytically destroys the target mRNA, resulting in gene silencing (Slicer). The messenger RNA stays uncleaved if the siRNA/mRNA duplex displays mismatches. When translational inhibition occurs, genes are silenced. This cartoon depicting RNA interference was adapted from the NCBI website: RNA Interference (RNAi) [4-9]. Functional studies of the mammalian genome can show how genetic changes cause changes in phenotype, and the Genetic Perturbation Platform (GPP), once called the RNA interference (RNAi) Platform, supports these investigations.

 

A helicase domain, two RNase III domains (RNase IIIa and RNase IIIb), a double-stranded RNA binding domain, a PAZ (Piwi-Argonaute-Zwille) domain, and a domain of an undetermined function (DUF283) are among the recognized functional domains [5, 6, 9]. Characterizing DICER and its domains in detail has proven difficult due to the protein's considerable size. Most domains' major activities and structural placements have been clarified by utilizing diverse study methods and homologs in other organisms (Figures 2-3).

 

 

 

 

Figure 2. Various dsRNA. Schematic depicting the characteristics and origins of various dsRNA substrates. There are two possible sources for genomic transcripts that produce double-stranded RNA (dsRNA): repeated transcripts in pathways that maintain genome integrity and genetic sequences in pathways that control gene expression (Adapted from an Open Access source: Zapletal D, Kubicek K, Svoboda P, Stefl R. Dicer structure and function: conserved and evolving features. EMBO Rep. 2023 Jul 5;24(7):e57215. doi: 10.15252/embr.202357215. Epub 2023 Jun 13. PMID: 37310138; PMCID: PMC10328071).

 

 

 

 

Figure 3. Substrate Cleaning. There are two ways to clean the substrate. During its processive mode, Dicer "feeds" its substrate by slicing lengthy dsRNA molecules in a series of sequential steps that are powered by the helicase domain's ATP activity. Dicer attaches a new substrate after performing a single cleavage when in distributive mode (Adapted from an Open Access source: Zapletal D, Kubicek K, Svoboda P, Stefl R. Dicer structure and function: conserved and evolving features. EMBO Rep. 2023 Jul 5;24(7):e57215. doi: 10.15252/embr.202357215. Epub 2023 Jun 13. PMID: 37310138; PMCID: PMC10328071).

 

 

 

The molecule contains two critical domains, such as the RNase IIIa and IIIb. The domains are the operational components responsible for cleaving dsRNA molecules. The helicase domain, which is located at the base of the protein, works in conjunction with the dsRNA binding domain (dsRBD) to identify and attach to the substrate. The domain, labeled as PAZ, is located at the N-terminus of the protein. It identifies the 5’ phosphate and two specific nucleotides of the substrate molecules. It  assists in their precise positioning. The function of DUF283 is currently ambiguous, although it has been proposed to play a task in the binding of RNA to other (protein) complexes and in enhancing the effectiveness of enzyme action [10-12].

The cleavage effect mediated by DICER produces two main products. They are micro-RNA (miRNA) and small interfering RNA (siRNA). MiRNA maturation commences in the nucleus by synthesizing a lengthy primary transcript RNA molecule (pri-miRNA). The pri-miRNA molecule is subsequently cleaved by an enzyme complex containing DROSHA, which is a member of the superfamily of RNase III. This cleavage results in a pre-miRNA molecule nearly 60 bases in length and possesses a hairpin structure. Exportin 5 facilitates the carriage of this molecule toward the cytoplasm from the nucleus, where it can be subsequently identified and cleaved by DICER to generate miRNA. On the other hand, siRNA molecules are generated through the process of DICER cleaving various double-stranded RNA precursors. The enzyme's structure and its domains, which have been labeled as the 'molecular ruler,' ensure that both miRNA and siRNA molecules are constantly 20-24 bases long.

Ago and the transactivating response RNA-binding protein (TRPB) are two more proteins that accompany the enzyme-substrate complex after DICER cleavage. The entire DICER-Ago-TRBP complex is commonly known as the 'RISC-loading complex,' responsible for loading the short double-stranded RNA products into another protein complex known as the RISC (RNA-induced silencing complex). The resulting combination of RISC and dsRNA is called miRISC or siRISC, depending on the specific type of tiny dsRNA present. This mechanism marks the initiation of the RNA interference (RNAi) pathway [13, 14].

The physiological function of DICER in humans is complex. DICER1 is ubiquitously expressed and is an essential housekeeping gene in physiological tissue development. Eight short RNA products produced by DICER play a role in several activities. Therefore, DICER is likely involved in more cellular functions than we know. DICER plays a function in miRNA processing and participates in DNA processing, remodeling of chromatin granule structure, and programmed cell death or apoptotic pathways (Table 1).

 

Table 1. Main DICER Roles in Pediatric and Youth Pathology

Time                                                                                                   Main Mechanism(s)

• Early Development of the Embryo                   Cell cycle kinetics, Caspase 3 activation and apoptosis
• Neural Progenitor-type Cells Proliferation Cell cycle kinetics
• Neuronal cell migration and differentiation             Cell cycle kinetics
• Astrocyte differentiation                   Cell cycle kinetics
• Limb morphogenesis                               Caspase 3 activation and apoptosis

(programmed cell death))

• Immunologic Competency                               B lymphocytic cellular function,
                                          Stress granule response
• Skin development                   Cell cycle kinetics

 

RNA interference

Post-transcriptional gene silencing, also known as RNA interference, is a mechanism that regulates the expression of genes that code for proteins. After forming the RISC-loading complex, it binds to the RISC, which is essentially the executor of the RNA interference paths. Upon activation of this complex, dsRNA undergoes separation. The passenger strand is eliminated and undergoes degradation, while the other strand persists in the RISC and functions as the guide strand. The RISC may now specifically attach to target RNA sequences, such as mRNA, and exert control over their translation or facilitate their breakdown. If the guide and the target have a perfect match, typically with siRNA, the Ago molecules (proteins) in the complex will utilize their endonuclease function to degrade the target, inhibiting translation. In the case of an flawed match, particularly with miRNA, the mRNA stays stable, but the translation process is inhibited. Perfect matching generally enables the selection of more precise objectives, whereas imperfect matching allows for a broader range of targets. The role of nuclear activities in repairing DNA harm is still under intense investigation. Contrary to earlier beliefs, new research has demonstrated that human DICER, thought to be only found in the cytoplasm, can be moved to the nucleus in reaction to specific stimuli. Typically, when DNA breaks into two strands, it activates the DICER protein through the ERK pathway, a member of the KRAS/MAPK system. This alteration of the protein facilitates translocation to the nucleus [10-12, 15]. Upon arrival, DICER undergoes phosphorylation, generating short RNA molecules from the regions neighboring the chromosomal breakage site. The precise mechanism beyond this juncture remains incompletely comprehended. However, investigations conducted on cells lacking The DICER1 study found that the DNA damage repair mechanism is less effective than before. The accumulation of DNA breaks in both strands and an increased susceptibility to radiation from ultraviolet and gamma rays are both outcomes of this.[16]. There are a number of other possible roles for this molecule in the nucleus, including repairing nucleotide excision, preserving heterochromatin structure, timing DNA replication, suppressing transposable elements, facilitating apoptosis, and autophagy [17].

 

Antiviral properties

DICER primarily acts on endogenous dsRNA but also plays a crucial part in antiviral pathways across several taxa, comprising invertebrates,  vertebrates, and plants.. It is established that in humans, it exhibits antiviral properties against some RNA viruses [10-22]. DICER can convert RNA of a virus into small RNA molecules called virus-derived small RNA molecular structures, or vsiRNAs, just like it does with endogenous dsRNA during normal physiological activity. Subsequently, these diminutive molecules can be employed comparably to miRNAs, wherein they bind and repress viral genes imperative for replication. Although the precise mechanism remains incompletely understood, research has demonstrated that DICER experiences downregulation in monocytes/macrophages during the infection of Human Immunodeficicy Virus 1 (HIV-1). Moreover, inhibiting DICER can enhance Adenovirus multiplication, further confirming this hypothesis [17]. DICER is also linked to the immune response against specific viruses, including Zika virus (a member of the virus family Flaviviridae primarily spread by mosquitoes) and SARS-CoV-2 (the etiologic agent for COVID-19 pandemics) [23-25]. There is a possibility that this system could be controlled by other viruses and ultimately generate pro-viral effects [26].

DICER1 in human disease and its pathobiology

The primary pathological function of DICER1 appears to be associated with the deregulation of miRNAs and, of course, the RNAi mechanism. DICER1 gene mutations can be classified as either germline or somatic. Germline mutations can arise at any location within the gene, often leading to loss of function. On the other hand, somatic mutations tend to occur in specific 'hotspots' [27-31]. As our understanding of the complete range of DICER1 function improves, we may uncover more nuanced roles in disease processes and, ideally, better comprehend the diseases themselves. Table 1 displays a compilation of specific disorders with which DICER1 has been associated. There is a concise overview of many disorders where dys-regulation (abnormal regulation) or molecular modification of DICER1 have been linked, along with their postulated causes and specific discoveries related to the disorders. DICER1 has been demonstrated to participate in a wide range of pathological diseases. Occasionally, DICER appears to operate correctly, yet it receives an atypical substrate. Trinucleotide repeat diseases include the formation of hairpin-like structures due to the presence of elongated chains of repeats. These complexes, like the standard substrate of the DICER molecule, undergo processing and aggregate in cells, resulting in cellular damage [21, 32]. Alternatively, the disease state is triggered in certain instances by the molecule's intracellular amounts, as observed in a specific kind of age-linked macular degeneration known as “geographic atrophy”. The absence of DICER in the retinal pigmented epithelium (RPE) is critical, because individuals with geographic atrophy may show the buildup of a harmful protein transcript, ultimately causing vision loss [33]. In fact, Saeki et al. [34] examined DICER and its co-RNase, DROSHA. They proposed that there may be a connection between particular genetic variations and autoimmune thyroid diseases such as Graves' disease and Hashimoto thyroiditis and the involvement of the NLRP3 inflammasome is still puzzling in a similar situation as seen in other diseases [35-40]. Their investigation also demonstrated a potential correlation between the levels of expression of these two proteins and the refractile/remission rate.

Furthermore, a longstanding correlation exists between a thyroidal disease, the multinodular goiter (MNG) and DICER1 mutations. Below the section on DICER1-related illnesses will contain further details. While the common assumption is that pediatric goiters are associated with DICER1 mutations, individuals with these mutations can be diagnosed with goiters in their forties or later, particularly if they have not suffered any other DICER1-related disorders. DICER has been identified as a potential cause in various diseases, for instance neuropsychiatric disorders, cardiovascular disease, and fertility problems [17]. The limited occurrences presented demonstrate the diverse ways DICER1 may influence or trigger disease, and much study is now underway.

Neoplasia refers to the abnormal growth and proliferation of cells, leading to tumor formation. A major obstacle in comprehending the role of DICER changes and dysregulation in human disease lies in the incomplete understanding of the subsequent pathways. Although numerous diseases have exhibited DICER-related alterations, there is a lack of consistent patterns, particularly concerning neoplasia [26]. As mentioned, DICER's impact on disease is mainly linked to the disruption of miRNAs, heading to alterations in overall genetic expression. Typically, cancerous growths have a decrease in the activity of small RNA molecules called miRNAs, and the absence of a properly functioning protein called DICER impairs the ability of cells to repair DNA damage [16]. DICER1 has been linked to the promotion of both benign and malignant neoplastic processes (tumors), and a hyperplastic and cystic phenotype characterizes the usual histological presentation, although this is not universally observed.

It has been suggested that DICER1 is a haplo-insufficient gene that suppresses tumor growth [41].  In haplo-insufficiency, one gene copy is either deleted or rendered inactive, leaving just one functioning copy that is inadequate to produce the required gene product and maintain normal function. However, its precise categorization remains a subject of ongoing discussion and remains controversial [26, 42]. The carcinogenesis associated with DICER1 has primarily been investigated in people with pathogenic germline gene mutations, resulting in the total loss of one allele of DICER1 (refer to the section on DICER1 syndrome). Abnormal characteristics occur because of a second (somatic) mutation. What is remarkable, though, is that the secondary mutation does not lead to the elimination of the second DICER1 copy (complete elimination of DICER1 will hinder cell growth and lead to cell death). Tumorigenesis requires a second mutation, typically a missense genetic mutation, which hampers the functionality of the DICER enzyme. Normally, the second impact strikes inside one of the five hotspot regions in the RNase IIIb region of the gene, resulting in a modification of the gene's capacity to break down substrate molecules. The outcome is an atypical combination of miRNAs within the cell, leading to alterations in gene expression [41]. The variation in the composition of miRNAs leads to variation in the resulting illness phenotype.

Although most DICER1-related thyroid lesions are non-cancerous, there have been observations of their presence in thyroid carcinomas, which can vary in their level of differentiation from well differentiated toward a very aggressive phenotype. Germline mutations in DICER1 that occur in both alleles are linked to a significantly higher risk of thyroid cancer, roughly 16 times more than the average risk. However, the risk seems modest in patients harboring a heterozygous germline genetic mutation. Oncogenesis is promoted in heterozygous mutations when there is a second promoting mutation in DICER1 or another gene. Similar genetic alterations have been observed in other well-known thyroid oncogenes, for instance BRAF and RAS.

Importantly, Canbrek et al. conducted a review and found that about one individual among fifty people (1.5–3.7%) harboring thyroid carcinomas exhibit a DICER1 mutation. However, the relevance of many of these mutations remains uncertain [43]. DICER1 has been found to share a regulatory transcription factor with TERT in vitro investigations conducted on thyroid cancer. These investigations demonstrated a correlation between reduced amounts of the transcription factor GABPA and lower levels of DICER, as well as an increase in tumor invasion, neoplastic cell proliferation, and overall tumor call viability. The precise mechanisms responsible for these discoveries remain uncertain [41, 42, 44].

DICER also has a crucial role in sporadic cancer instances. In instances when heredity is not a factor, somatic genetic mutations arise in the identical hotspot region of the RNase IIIb gene, leading to modified protein functionality [45]. The initial DICER1 genetic mutation can then be linked to a secondary genetic mutation, which can be either another somatic genetic mutation, a “mosaic landscape”, or a distinct germline genetic mutation to facilitate tumor development [43]. Phosphorylation of DICER in certain animal studies was found to be linked with elevated levels of tissue and lympho-vascular invasion. When combined with other genetic mutations, for example in KRAS and p53, mice exhibited a more comprehensive range of neoplastic processes, which were also more likely to spread to numerous organs. Several investigations involving the suppression of DICER1 gene expression demonstrated tumors developing heightened invasiveness and faster growth rates [15].

Table 2. Dysregulation or molecular alteration of DICER1 connected in Human Neoplastic Diseases [17].

Tumor                                    DICER1 Alteration (Red=down-regulation/Green=up-regulation)

NSCLC
CRC
Prostate Ca
Breast Ca
ALL
Ovary Ca
Ovary Ca
Thyroid Ca
Thyroid Ca

Notes: Reduced expression is red, while increased expression is green. ALL, acute lymphoblastic leukemia; NSCLC, non-small cell lung carcinoma; CRC, colorectal carcinoma; In Trinucleotide repeat disorders, such as Huntington’s disease, Myotonic dystrophy type 1, and Fragile X syndrome both normal and increased expression has been recorded. Autoimmune disorders (e.g., ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, and autoimmune thyroid disease) are overwhelmingly reporting a reduced expression apart from psoriasis. Other diseases, such as chronic stress, depression, Parkinson’s disease, atherosclerosis, have shown a reduced expression.

 

Disorders associated with DICER1 gene mutations

Before discovering and classifying RNA interference in detail and DICER, researchers were investigating cases of autosomal dominant (AD) inherited (familial) multi-nodular goiter (MNG). These family units had an abnormally high prevalence of individuals with strumae (goiters) that typically occurred throughout childhood or youth (early adulthood). It is now understood that benign thyroid lesions are the most prevalent indication of inherited DICER1 mutations [27, 28, 46-48]. Delahunt and colleagues documented the initial familial correlation between pleuropulmonary blastoma and cystic nephroma in 1993 [49]. Subsequently, a greater number of tumors, both non-cancerous and cancerous, have been linked to inherited DICER1 defects, including disorders characterized by mosaicism, with varying degrees of manifestation.

Similar to other hereditary neoplasia-related conditions, individuals with this ailment are typically diagnosed with lesions at a younger age and usually require ongoing monitoring. For individuals with a specific set of genetic mutations known as "mosaicism for RNase IIIb domain hotspot mutations," rigorous surveillance protocols are advised. This group typically exhibits a higher degree of genetic penetrance, with the manifestation of the disease occurring at a younger age and a broader range of observable characteristics. Currently, there have been no known specific associations between changes in the DICER1 gene and observable characteristics [50].

DICER1 syndrome

DICER1 syndrome, which stands for DICER1-related pleuropulmonary blastoma cancer predisposition syndrome, is passed down through families in a reduced-penetrance autosomal dominant fashion. Although individuals with this genetic syndrome have an elevated risk of acquiring tumors, particularly thyroid carcinoma, most patients can expect to lead healthy lives. The frequency of germline loss of function (LOF) mutations in the DICER1 gene is roughly 1 in 10,600, with merely a tiny fraction of these newly occurring [51]. Individuals affected by DICER1 syndrome experience the growth of both non-cancerous and cancerous tumors, with the majority of these tumors appearing before reaching the age of 40.

Most often, germline mutations manifest as “nonsense, frameshift, or splice site” genetic mutations, as well as minor deletions and insertions. These mutations might potentially occur at any location within the gene. As mentioned in the previous segment on neoplasia, it has been proposed that the development of tumors in DICER1 syndrome is caused by the inactivation of one allele of the DICER1 gene in the germline, followed by the acquisition of a somatic mutation in the lasting allele. This mutation modifies the exons that contain the RNase IIIa and IIIb domains, leading to a decrease in the production of miRNAs. The thyroid, lungs, kidneys, and ovaries are the primary organs most impacted by DICER1 syndrome. The most prevalent manifestation in general is MNG. By the age of 20, 32% of females and 13% of males with DICER1 syndrome will have acquired MNG. These percentages rise to 40%, and even, 17% respectively by age 40 years. Pleuropulmonary blastoma is commonly regarded as the most frequent cancerous occurrence, occurring in approximately 30-40% of individuals with DICER1 disease. This neoplasm primarily affects young children and is usually diagnosed before the age of six years. It can either manifest as benign cystic lesions (type Ir) or as an aggressive malignancy of mesenchymal type (sarcomatoid neoplasm) (type III). The prevalent diagnoses in the ovaries and kidneys are Sertoli-Leydig cell tumor of the ovaries and cystic nephroma of the kidney, respectively.

 

 

Figure 2. CT Imaging and Macroscopic View of Pleuropulmonary Blastoma. In a), CT scan of a 21 year old female patient with a pleuropulomonary blastoma causing a mediastinal shift. In b), the figure shows the resected pleuropulmonary blastoma just after surgery. No copyright issue. The images come from the personal archive of Dr. F. Minervini.

 

Figure 3. Microphotographs of Pleuropulmonary Blastoma. A heterogeneous solid tumor (a) abutting (arrow) the fibrotic pleura showing on higher magnification blastema-like area with hyperchromatic nuclei, high nucleus to cytoplasm ratio and mitotic bodies (b). In this case, foci of anaplastic cells with pleomorphic nuclei and mitotic figures were also observed (inset). Hematoxylin and eosin staining, scale bar embedded in the microphotographs. No copyright issue. The images come from the personal archive of Dr. C. Sergi.

 

 

Additional non-tumoral abnormalities have also been documented in individuals with DICER1 syndrome. The mentioned characteristics encompass macrocephaly, visual abnormalities, kidney and renal collecting system structural anomalies, and odontodysplasia or dental deformities [52, 53]. Although specific abnormalities associated with DICER1 syndrome, such as thyroid lesions, are relatively common in the general community, people with this syndrome tend to develop these diseases at a earlier age than the usual people. Pediatric populations have been observed to exhibit poorly differentiated thyroid cancer, which is usually found in older individuals and is related to DICER1 mutations [54]. Most of the remaining malignancies associated with DICER1 syndrome are uncommon, which theoretically facilitates the discovery of patients and families for screening purposes. Schultz et al. present a comprehensive overview of these tumors, along with recommendations for determining the diagnosis of DICER1 syndrome [55]. Other rare tumors have been described in individuals harboring DICER1 syndrome [56]. They include cranial (central nervous system) and extracranial (extra-central nervous system) neoplasms. Cranial neoplasms include medulloblastoma, ciliary body medulloepithelioma, pineoblastoma, and pituitary blastoma. Extra-cranial neoplasms include embryonal rhabdomyosarcoma, botryoid rhabdomyosarcoma of the cervix, gynandroblastoma of the ovary, gonadal granulosa cell tumor, gonadal choriocarcinoma, differentiated thyroid carcinoma, nasal chondromesenchymal hamartoma, exocrine and endocrine pancreatic carcinoma, ovarian fibrosarcoma, renal sarcoma and peritoneal sarcoma. All these neoplastic processes arise from a germline mutation in the DICER1 gene, leading to a predisposition for developing these tumors in different organs like the lungs, kidneys, ovaries, thyroid, pancreas, and central nervous system. Their pathology does not differ from tumors originating in individuals without DICER1 gene mutation.

 

GLOW syndrome.

The term GLOW syndrome stands for Global developmental delay (psychological/psychometric delay), Lung cysts, Overgrowth (somatic), and Wilms tumor or nephroblastoma, a disontogenetic neoplasm, which has been associated to nephrogenic rests [57] (Figure 4). Most recently, Wilms tumor has been associated to an exposure to parental contact to pesticides, especially in household settings and with organophosphate compounds [58].

 

Figure 4. DICER1 associated lung cysts in an asymptomatic 22-year-old male patient. No copyright issue. The image comes from the personal archive of Dr. F. Minervini.

 

Glow syndrome was initially introduced by Klein et al. in 2014, and then further explored [42, 59-62]. The authors showcased two instances of children exhibiting global developmental delay (psychologic/psychometric) and somatic overgrowth disorders in which mosaic DICER1 expression was seen. The tissue expression level varied between 21% and 47%, provisional on the patient and the kind of tissue. Subsequently, both pediatric patients experienced the development of pulmonary cysts and Wilms tumors, exhibiting a phenotype comparable to other disorders characterized by excessive tissue growth and a vulnerability to malignancies. Lung cysts should be a red flag indicating that the pediatrician should think about abnormal development at first place. Nephroblastoma may usually occur in the 2^nd infancy or later childhood [56].

In these cases, genetic research revealed a mosaic pattern of a newly occurring DICER1 mutation. This mutation was found in specific locations inside the RNase IIIb domain, which is recognized to be associated with “metal binding”. In both cases, the Wilms tumors and intact kidney tissue had a greater mutation prevalence than blood cells. Upon examination of the Wilms tumor in one patient, it was found that additional somatic mutations were explicitly present in the tumor tissue. However, the same mutations were not observed in the other case. Overall, GLOW syndrome should be considered as one of the DICER1-related illnesses. They postulate that specific genetic mutations in DICER1, particularly those that affect “metal-binding” sites in the Rnase IIIb domain, may enable DICER1 to act as an oncogene. No definitive mechanism exists to elucidate the precise locations implicated (or not implicated) in GLOW syndrome [42, 59-62].

Assessing for DICER1 mutations: timing and methodology

Although DICER expression appears widespread, the reasons for the differential impact on various organs and tissues remain unknown. There is no evident connection between the various cancerous growths other than a casual preference for a less developed or embryonic differentiation. However, it is essential to mention that many non-neoplastic disorders linked to alterations in DICER expression are recognized to exhibit a wide range of anatomical indications and symptoms. Specific lesions exhibit a strong correlation with DICER1 mutations, thereby necessitating testing. Furthermore, it is crucial to assess when pediatric patients exhibit typical thyroid lesions commonly seen in adults, such as MNG or poorly differentiated thyroid cancer [28, 46, 47, 63, 64]. Germline DICER1 mutations predominantly manifest as deletions or duplications/insertions, with occasional occurrences of translocations, but infrequently [17]. Copy number variations are the most frequent genetic changes observed in sporadic malignancies. These modifications often lead to increased expression of the DICER1 protein.

Additionally, missense mutations are very commonly found. Given the very limited occurrence of malignant tumors, identifying a disease-causing mutation in the body's cells should at least lead to suspicion of a mutation in the reproductive cells [65].

When assessing an individualfor DICER1 syndrome, molecular genetic testing may be specifically targeted depending on the patient's profile. If the symptoms match those typically seen in DICER1 syndrome, conducting single testing or employing a targeted genetic panel may be beneficial. If the phenotype is less clear-cut, genetic research may be required.

As a general guideline, it may be critical initiating the search by focusing on missense and nonsense genetic mutations as well as insertions/deletions, , and splice site variants. If no anomaly is detected, it may be essential to do tests for more extensive deletions or duplications. Utilizing an inherited cancer multigene-based panel is an effective strategy to optimize the accurate detection of cancer genes and minimize expenses and potential complications, such as ambiguous genetic variations. Around one in 10 (10%) of patients with a new DICER1 mutation will exhibit somatic DICER1 mosaicism. In such instances, it may be necessary to conduct tests on various tissue types to definitively establish the existence of a harmful mutation. Furthermore, because DICER1-related sickness is very recent, it is typical to come across variations of unknown significance when evaluating these patients. De Kock, Wu, and Foulkes propose an advisable method for handling these circumstances [50].

DICER and therapies

Currently, no specific therapies for cancers and illnesses include mutations in the DICER1 gene. The gene's involvement in numerous disease states makes it an appealing target. However, the challenge is finding its therapeutic potential without risking substantial side effects until the complete range of its functions is uncovered. Certain medications now in use have demonstrated the capacity to enhance DICER1 activity. Metformin has been shown to modify the distribution of DICER, resulting in an elevation of its cytoplasmic concentrations, as observed in both mice and human research. Enoxacin, a type of antibiotic called fluoroquinolone, has been found to enhance the activity of DICER [66]. In addition to antibiotic properties, enoxacin has also been demonstrated to successfully inhibit the growth of multiple neoplastic cells [67]. Fascinatingly, enoxacin has been conveyed to have a low incidence rate of toxicity due to its ability to selectively prevent the growth of neoplastic cells, while leaving nonneoplastic cells untouched. The enoxacin adjuvant therapy could potentially have a protective effect on the brain in Parkinson's disease. The role of epigallocathechin gallate is particularly interesting and under intense investigation, particularly considering the protective effect of green tea against neurodegenerative diseases [68]. Interestingly, enoxacin and epigallocatechin gallate accomplish synergistically to inhibit the growth of cervical neoplastic cells [69]. Additionally, interferon-beta1 has been shown to raise the levels of DICER in the blood of certain people with multiple sclerosis who have had positive clinical outcomes [17]. The potential intersection with TERT pathways15 may also hold valuable therapeutic implications. However, further inquiry is required, as said earlier.

We are deeply grateful to you and all reviewers.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article provides a comprehensive overview of the DICER1 gene, its functions, and its implications in human health and disease. Here are some key points of commentary on the content: The article effectively highlights the essential role of DICER1 in RNA processing and its involvement in critical biological processes such as RNA interference, DNA damage repair, and antiviral responses. This underscores the gene's significance in maintaining cellular homeostasis and responding to environmental challenges. The discussion of DICER1's expression across various organ systems and its importance in normal human development is particularly noteworthy. The connection made between DICER1 mutations and specific clinical syndromes, such as DICER1 syndrome and GLOW syndrome, emphasizes the gene's relevance in pediatric oncology and genetic disorders. This aspect of the article could be expanded by discussing the mechanisms through which DICER1 mutations contribute to tumorigenesis.

Improve abstract and conclusion:  it’s essential to enhance clarity, cohesiveness, and impact while summarizing the key points effectively.

Summarize Key Points:

Highlight Clinical Significance:

Discuss Future Directions:

Incorporate figures, tables, or infographics that summarize key points, illustrate pathways, or compare DICER1 with other similar genes. Visuals can enhance understanding and retention of information.

------------------------------------------------------------------

the role of the DICER1 gene in human health and disease, and how do its mutations contribute to specific clinical syndromes and disorders to be relevant to the field, but not necessarily original. The DICER1 gene and its functions have been well-studied in the past, and its involvement in RNA processing, DNA damage repair, and antiviral responses is already established in the scientific literature.

However, the topic is still relevant because it provides a comprehensive overview of the gene's functions and its implications in human health and disease. The discussion of DICER1's expression across various organ systems and its importance in normal human development is also a valuable contribution to the field.

The topic does address a specific gap in the field, which is the need for a comprehensive review of the DICER1 gene's functions and its role in human disease. The commentary suggests that the article could be expanded to discuss the mechanisms through which DICER1 mutations contribute to tumorigenesis, which indicates that there is still a need for further research in this area.

Overall, while the topic may not be entirely original, it is still relevant and addresses a specific gap in the field by providing a comprehensive overview of the DICER1 gene's functions and its implications in human health and disease.

 

1. Comprehensive overview: The article provides a comprehensive overview of the DICER1 gene's functions, its involvement in critical biological processes, and its implications in human health and disease. This comprehensive approach may not be present in other published materials.

2. Integration of multiple aspects: The article discusses the DICER1 gene's expression across various organ systems, its importance in normal human development, and its connection to specific clinical syndromes, such as DICER1 syndrome and GLOW syndrome. This integration of multiple aspects of the gene's functions and implications may not be present in other published materials.

3. Pediatric oncology and genetic disorders perspective: The article's focus on the relevance of the DICER1 gene in pediatric oncology and genetic disorders may provide a unique perspective on the subject area.

4. Potential for mechanistic insights: The commentary suggests that the article could be expanded to discuss the mechanisms through which DICER1 mutations contribute to tumorigenesis. If the article provides new insights into these mechanisms, it would add significant value to the subject area.

Overall, the article appears to add a comprehensive and integrated perspective on the DICER1 gene's functions and implications in human health and disease, with a focus on pediatric oncology and genetic disorders.

 

The article effectively highlights the essential role of the DICER1 gene in RNA processing and its involvement in critical biological processes, and it discusses the gene's expression across various organ systems and its importance in normal human development.

The conclusions also address the main question posed, which is the role of the DICER1 gene in human health and disease. The article's discussion of the connection between DICER1 mutations and specific clinical syndromes, such as DICER1 syndrome and GLOW syndrome, provides evidence of the gene's relevance in pediatric oncology and genetic disorders.

However, the commentary suggests that the article could be improved by providing more mechanistic insights into how DICER1 mutations contribute to tumorigenesis. This suggests that the conclusions may not fully address the main question posed, as they do not provide a complete understanding of the underlying mechanisms.

Additionally, the commentary does not provide information on the article's limitations, potential biases, or alternative explanations for the findings. Therefore, it is unclear whether the conclusions are overly broad or whether they are supported by a thorough consideration of the evidence.

Overall, the conclusions appear to be generally consistent with the evidence and arguments presented, but they may not fully address the main question posed due to the lack of mechanistic insights. Further discussion of the limitations and potential biases of the study would strengthen the conclusions and provide a more comprehensive understanding of the DICER1 gene's role in human health and disease.

 

To fully address the main question posed, the conclusions could be strengthened by:

1. Providing more mechanistic insights into how DICER1 mutations contribute to tumorigenesis.

2. Discussing the limitations and potential biases of the study.

3. Considering alternative explanations for the findings.

4. Providing a more comprehensive review of the existing literature on the DICER1 gene and its role in human health and disease.

 

 

Author Response

The article provides a comprehensive overview of the DICER1 gene, its functions, and its implications in human health and disease. Here are some key points of commentary on the content: The article effectively highlights the essential role of DICER1 in RNA processing and its involvement in critical biological processes such as RNA interference, DNA damage repair, and antiviral responses. This underscores the gene's significance in maintaining cellular homeostasis and responding to environmental challenges. The discussion of DICER1's expression across various organ systems and its importance in normal human development is particularly noteworthy. The connection made between DICER1 mutations and specific clinical syndromes, such as DICER1 syndrome and GLOW syndrome, emphasizes the gene's relevance in pediatric oncology and genetic disorders. This aspect of the article could be expanded by discussing the mechanisms through which DICER1 mutations contribute to tumorigenesis.

Improve abstract and conclusion:  it’s essential to enhance clarity, cohesiveness, and impact while summarizing the key points effectively.

Summarize Key Points:

Highlight Clinical Significance:

Discuss Future Directions:

Incorporate figures, tables, or infographics that summarize key points, illustrate pathways, or compare DICER1 with other similar genes. Visuals can enhance understanding and retention of information.

------------------------------------------------------------------

the role of the DICER1 gene in human health and disease, and how do its mutations contribute to specific clinical syndromes and disorders to be relevant to the field, but not necessarily original. The DICER1 gene and its functions have been well-studied in the past, and its involvement in RNA processing, DNA damage repair, and antiviral responses is already established in the scientific literature.

However, the topic is still relevant because it provides a comprehensive overview of the gene's functions and its implications in human health and disease. The discussion of DICER1's expression across various organ systems and its importance in normal human development is also a valuable contribution to the field.

The topic does address a specific gap in the field, which is the need for a comprehensive review of the DICER1 gene's functions and its role in human disease. The commentary suggests that the article could be expanded to discuss the mechanisms through which DICER1 mutations contribute to tumorigenesis, which indicates that there is still a need for further research in this area.

Overall, while the topic may not be entirely original, it is still relevant and addresses a specific gap in the field by providing a comprehensive overview of the DICER1 gene's functions and its implications in human health and disease.

 

1. Comprehensive overview: The article provides a comprehensive overview of the DICER1 gene's functions, its involvement in critical biological processes, and its implications in human health and disease. This comprehensive approach may not be present in other published materials.
   
   2. Integration of multiple aspects: The article discusses the DICER1 gene's expression across various organ systems, its importance in normal human development, and its connection to specific clinical syndromes, such as DICER1 syndrome and GLOW syndrome. This integration of multiple aspects of the gene's functions and implications may not be present in other published materials.
   
   3. Pediatric oncology and genetic disorders perspective: The article's focus on the relevance of the DICER1 gene in pediatric oncology and genetic disorders may provide a unique perspective on the subject area.
   
   4. Potential for mechanistic insights: The commentary suggests that the article could be expanded to discuss the mechanisms through which DICER1 mutations contribute to tumorigenesis. If the article provides new insights into these mechanisms, it would add significant value to the subject area.
   
   Overall, the article appears to add a comprehensive and integrated perspective on the DICER1 gene's functions and implications in human health and disease, with a focus on pediatric oncology and genetic disorders.

 

The article effectively highlights the essential role of the DICER1 gene in RNA processing and its involvement in critical biological processes, and it discusses the gene's expression across various organ systems and its importance in normal human development.

The conclusions also address the main question posed, which is the role of the DICER1 gene in human health and disease. The article's discussion of the connection between DICER1 mutations and specific clinical syndromes, such as DICER1 syndrome and GLOW syndrome, provides evidence of the gene's relevance in pediatric oncology and genetic disorders.

However, the commentary suggests that the article could be improved by providing more mechanistic insights into how DICER1 mutations contribute to tumorigenesis. This suggests that the conclusions may not fully address the main question posed, as they do not provide a complete understanding of the underlying mechanisms.

Additionally, the commentary does not provide information on the article's limitations, potential biases, or alternative explanations for the findings. Therefore, it is unclear whether the conclusions are overly broad or whether they are supported by a thorough consideration of the evidence.

Overall, the conclusions appear to be generally consistent with the evidence and arguments presented, but they may not fully address the main question posed due to the lack of mechanistic insights. Further discussion of the limitations and potential biases of the study would strengthen the conclusions and provide a more comprehensive understanding of the DICER1 gene's role in human health and disease.

 

To fully address the main question posed, the conclusions could be strengthened by:

1. Providing more mechanistic insights into how DICER1 mutations contribute to tumorigenesis.

2. Discussing the limitations and potential biases of the study.

3. Considering alternative explanations for the findings.

4. Providing a more comprehensive review of the existing literature on the DICER1 gene and its role in human health and disease.

Thank you very much for your comments and suggestions.

We have extensively revised the manuscript; pay attention to adding more references, mechanistic insights, limitations, strengths, and future directions. We sincerely hope to have brought forward a thorough review on this topic. The article remains an expression of the experience of the authors and may not include everything like a book. Nevertheless, we think that the current manuscript is quite comprehensive.

1. Providing more mechanistic insights into how DICER1 mutations contribute to tumorigenesis.
   Please see the section between line number 539-594: 

   The Two-Hit Hypothesis: Insights into Molecular Mechanisms of DICER1 Mutations

   The two-hit concept, which was formally postulated by Knudson in 1971 [70, 71], was initially proposed in 1953. The theory proposes that abnormalities can only manifest when two distinct mutations occur in each allele, and that tumors cannot be caused by a single mutation in any one allele. The average person inherits a single mutation, which poses little health risk on its own. But it is also possible for two mutations to work together to cause cancer. Several instances of tumor suppressor gene deactivation can be explained by the two-hit concept [72, 73]. It has been established that DICER1 syndrome is an inherited, haploinsufficient autosomal dominant illness [74, 75]. Evidence from multiple DICER1 syndrome instances supports this hypothesized mechanism. These examples included DICER1 syndrome symptoms in people with a single apparent germline mutation. In contrast, new research shows that DICER1 syndrome individuals had a somatic mutation in the second DICER1 gene allele in addition to inherited alterations in the first allele. When discussing DICER1 mutations and Dicer's function as a tumor suppressor gene, the two-hit hypothesis is relevant. The DICER1 gene's RNase IIIb domain has second-hit somatic mutations. According to research including three Wilms' tumor patients, the two-hit hypothesis was relevant to the development of Wilms' tumor in patients with DICER1 syndrome [76]. Screening for somatic DICER1 mutations revealed that the patients had germline mutations in the gene, and further testing revealed that the patients had somatic mutations in the RNase IIIb region on the second allele. This discovery also brings attention to the fact that Wilms tumor patients often have mutations in the RNase IIIb domain of the DICER1 somatic gene. There are genetic hotspots for somatic mutations within the DICER1 gene inside the areas encoding the RNase III domains [77, 78]. The second mutation in pleuropulmonary blastomas often occurs in the RNase IIIb domain and is part of a biallelic DICER1 mutation [79]. In research involving eleven patients with spontaneous pleuropulmonary blastomas and DICER1 gene mutations, eight of the patients had biallelic DICER1 gene mutations, with one mutation located in the RNase IIIb domain. An ovarian fibrosarcoma tumor from a 9-year-old girl was found to have biallelic DICER1 mutations. The tumor also had a second point mutation, which caused a substitution at amino acid position 1813 within the RNase IIIb domain (p.E1813G) [80]. The tumor also had a germline single base insertion in the DICER1 gene, which caused a frameshift and premature stop codon.

    

   In another instance, a female patient who was 14 months old at the time of diagnosis had pleuropulmonary blastoma in addition to a cystic nephroma that had been resected 11 months earlier [81]. The DICER1 gene was found to be compromised in both alleles after mutation analysis was performed on peripheral blood and accessible tissue. The DNA sample from the cystic nephroma included a missense heterozygous somatic mutation (c.5425G>A; p.G1809R), and there was also a germline truncating mutation (c.5347C>T; p.Q1783*) in exon 24, which codes for the RNase IIIb domain, in the peripheral blood. The patient's maternal lineage was found to carry this genetic mutation. A female cousin of the patient, who is 21 years old, was also discovered to carry the germline mutation. She had undergone treatment for embryonal rhabdomyosarcoma when she was 14 years old and multimodal goiter when she was 20 years old. The patient's embryonal rhabdomyosarcoma (c.5428G>C; p.D1810H) was found to have a novel missense heterozygous somatic mutation. This instance shows that the mechanism of DICER1 syndrome is not due to haploinsufficiency but rather to biallelic mutations in the DICER1 alleles. Second, somatic, hot-spot alterations in the RNase IIIb domain were the subject of Brenneman and colleagues' [82] discussion of biallelic DICER1 gene mutations. The mutation status of DICER1 alleles was determined in a cohort of patients diagnosed with pleuropulmonary blastoma. It is possible that the rate-limiting phase in the pathophysiology of DICER1 syndrome involves mutations inside the RNase IIIb domain, which could constitute hotspot mutations [83]. Patients typically had DICER1 germline mutations that caused a loss of function, and while RNase IIIb hotspot mutations were uncommon outside of tumors, they were more common inside them. Because Dicer is involved in DNA replication and repair, patients with RNase IIIb mutations may also be at increased risk for other mutations.

2.  

   2. Discussing the limitations and potential biases of the study

Please see between line numbers 597 and 625

Limitations

Research on DICER1's function in exocrine pancreatic cancer and endocrine pancreatic cancer is limited [84, 85]. The process of acinar-to-ductal metaplasia (ADM) and epithelial-to-mesenchymal transition (EMT) in pancreatic acinar cells was shown to begin in mice with homozygous knockout of Dicer1 [86]. The onset of pancreatic intraepithelial neoplasms (PanIN) was accelerated in a study by Wang et al. (2014) [86] and by Eser et al. (2014) [87], who found that oncogenic Kras, a small GTPase, promotes pancreatic neoplasia when activated simultaneously with heterozygous deletion of Dicer1. Kras activation in conjunction with Dicer1 homozygosity resulted in increased ADM formation but had no effect on PanIN initiation rate [86]. They also discovered that acinar cell regeneration was allowed in the absence of Kras activation after extensive Dicer1 deletion. Despite the importance of this discovery, it is not completely ruled out that other variables, like a population of progenitor cells or transdifferentiated cells, may have had a role in the pancreatic cell regeneration [86, 88]. These results suggest that the exocrine pancreas tumor growth could be dose-dependent for DICER1.

To confirm the role of DICER1 in pancreatic cancer, additional research is needed to address potential technical limitations, such as the fact that Dicer1fl/fl animals retain one functional Dicer1 allele or the difficulty in achieving complete loss of Dicer [86]. Wang et al. (2012) [89] observed the effects of systematically knocking down DICER1 expression in a number of pancreatic cancer cell lines (BxPC-1, Capan-2, Panc-1) on the expression of oncogenes and tumor suppressor genes. In all three cell lines, knocking out Dicer1 caused variations in the expression profiles of the Kras oncogene as well as the tumor suppressors p53 and PTEN. A decrease in Dicer1 expression slowed the development of BxPC-1 tumors but increased that of Panc-1 tumors, with no discernible impact on Capan-1 tumors. It is essential to have tailored treatments for pancreatic cancer because distinct subtypes are caused by distinct molecular pathways [89]. We do not yet know whether these discoveries will lead to endocrine pancreas tumors. Even if there aren't many pancreatic tumors that start in the endocrine system, we still need to undertake additional research to find out if DICER1 is a good therapeutic target.

 

3. Considering alternative explanations for the findings.

This is a critical question and we added a section on future directions to answer a lot of questions about DICER1. Please see between line numbers 627 and 676 the section:

Future Directions

Since its recent discovery, our understanding of DICER1 syndrome has expanded greatly; yet, there is a pressing need for additional research into potential treatments and management strategies. Model animal studies on Dicer have sought to identify effective ways of increasing levels of Dicer and related proteins to counteract the consequences of DICER1 syndrome, since this insufficiency is caused by DICER1 mutations. Blandino and colleagues performed one such trial in which they administered metformin to diabetic mice, which decreased the cancer incidence [90]. Mice given metformin had a decrease in tumor growth, an increase in DICER1 gene expression, and an elevation of microRNAs. A related and more recent study corroborated these findings; it demonstrated that metformin increases Dicer levels in both mice and humans by changing the location of AUF1, a protein that binds to DICER1 mRNA and down-regulates DICER1, therefore increasing the stability of DICER1 mRNA [91, 92]. Metformin and similar compounds may help patients with a single functional DICER1 allele, as it is possible to target and upregulate this single allele to achieve increased Dicer protein production. However, these treatments may not work for patients with biallelic DICER1 mutations. A nonsense mutation in the DICER1 gene has been found in certain families; this mutation causes nonsense-mediated RNA degradation, shortened proteins, or stop codons in the coding sequence.

Antibiotics can restore transcription and translation of otherwise unintelligible sequences by encouraging the read-through of comparable premature stop codons, which causes tumors to develop in both cancerous and non-cancerous forms [93]. Human cells with known nonsense mutations were treated with the pharmaceutical medicine Ataluren, which is used to treat genetic problems, in one study to demonstrate this new technique. All three nonsense codons in the mutant alleles were read-through by Ataluren [94]. An updated study expanded on these results by changing the amount and placement of fluorines in Ataluren, which improved the read-through capability of stop codons in nonsense mutations [95]. Patients with DICER1 syndrome caused by nonsense mutations similar to those already studied may benefit from using Ataluren or comparable chemicals, while no research has been done on DICER1 nonsense mutations using these medications. Although there has been a lot of focus on treatments for pleuropulmonary blastoma and cystic nephroma, further research is needed to understand the genetic abnormalities and acquired somatic mutations that cause these aberrant forms.

More questions may need to be answered. In order to generate short RNAs of specific lengths, how do noncanonical Dicers, like Dictyostelium's DrnA and DrnB, identify and cleave substrates? Do protists have any additional Dicer architectures? Dicer is the only gene in C. elegans that can encode both microRNAs and small interfering RNAs.  What is the difference or similarity in the recognition and cleavage of lengthy dsRNA and pre-miRNA substrates? In other words, does Dicer mainly interact with pre-miRNAs via the PAZ domain, or does it recognize and load long dsRNA into the enzyme through the helicase domain? This question could be answered by having cryo-electron microscopy structures of C. elegans Dicer with both substrates.

As a result of binding Dicer, how does AGO choose 5p/3p miRNAs? In several model organisms, the exact mechanism by which AGO binds cleavage products and interacts with Dicer is yet unknown. Thermodynamic sensing encourages main strand selection and loading after Dicer releases a cleavage product. Nevertheless, further structural understanding of the RISC loading complex's organization and the loading process is required.

Is PACT, the mammalian TARBP2 paralog, physiologically significant? Is it serving a particular purpose?

To what extent are its activity and substrate specificity controlled by other Dicer-binding partners?

4. Providing a more comprehensive review of the existing literature on the DICER1 gene and its role in human health and disease.

We comprehensively review the literature with the most frequent tumors that a pediatric surgeon and pediatric pathologist can encounter in practice, but we listed also other tumors, which may be more frequent at older age. Please see the section at 435 and 447 for a list of neoplasms associated with DICER1 syndrome.

The illustrative material comes directly from our practice, and our paper avoided introducing neoplasms without DICER1 mutation.

We are deeply grateful to you and all reviewers.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

 This manuscript provides an overview of DICER1 function and highlights its role in DICER1 mutation-related human diseases. While the topic is important and relevant, the manuscript has some notable shortcomings that require attention, particularly in referencing key information.

Specific Comments

Page 2

Species Name Formatting:

The sentence: “Two genes encoding enzymes were discovered in D. melanogaster in 2001 [1].”

The species name D. melanogaster should be italicized.

Missing References:

The statement: “This set of proteins is ubiquitously present in eukaryotes, and although prokaryotes lack an analogous protein, they possess proteins that operate in comparable pathways.”

Please provide a reference to support this information.

 

Similarly, the sentence: “In general, the primary role of RNase III proteins is to identify and split apart double-stranded RNA molecules.”

Also requires a citation to substantiate the claim.

 

Figure 1

The caption for Figure 1 contains significant information about the function of DICER1. However, it does not include any references. Please provide appropriate sources to validate the data and claims presented in the figure.

General Recommendation

I strongly recommend that the authors carefully review the manuscript to ensure all key statements, claims, and figures are supported by appropriate references. This is a critical aspect of review articles, as it enhances the manuscript's scientific rigor and aids readers in locating relevant studies for further exploration.

Author Response

This manuscript provides an overview of DICER1 function and highlights its role in DICER1 mutation-related human diseases. While the topic is important and relevant, the manuscript has some notable shortcomings that require attention, particularly in referencing key information.

Specific Comments

Page 2

Species Name Formatting:

The sentence: “Two genes encoding enzymes were discovered in D. melanogaster in 2001 [1].”

The species name D. melanogaster should be italicized. 

The name was italicized. Thank you.

Missing References:

The statement: “This set of proteins is ubiquitously present in eukaryotes, and although prokaryotes lack an analogous protein, they possess proteins that operate in comparable pathways.”

Please provide a reference to support this information.

A reference [3] was added at line number 64. Thank you

 

Similarly, the sentence: “In general, the primary role of RNase III proteins is to identify and split apart double-stranded RNA molecules.”

Also requires a citation to substantiate the claim.

The line number 67 includes now the reference [3]. Thank you.

 

Figure 1

The caption for Figure 1 contains significant information about the function of DICER1. However, it does not include any references. Please provide appropriate sources to validate the data and claims presented in the figure.

 

Thank you. This cartoon depicting RNA interference was adapted from the NCBI website: RNA Interference (RNAi) [4-9]. 

General Recommendation

I strongly recommend that the authors carefully review the manuscript to ensure all key statements, claims, and figures are supported by appropriate references. This is a critical aspect of review articles, as it enhances the manuscript's scientific rigor and aids readers in locating relevant studies for further exploration.

All references have been double-checked and added more sections and more references as required. 

Thank you very much for your comments and suggestions. We revised the manuscript extensively and adopted additional references. New sections including limitations and future direction have been added to the manuscript.

We are deeply grateful to you and all reviewers.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

In this review, the authors illustrated DICER1's functions and how they relate to both cancer-causing and non-cancerous illnesses. DICER1, is a gene located on chromosome 14q32.13. It contains 27 exons linking the two enzyme domains. DICER1 is a vital housekeeping gene that encodes a multi-domain enzyme in small RNA processing. This enzyme functions in several pathways, including RNA interference, DNA damage repair, and antiviral responses. At the protein level, the endoribonuclease DICER is also involved in several human diseases, of which the pleuropulmonary blastoma is probably the most egregious entity.

Overall, this manuscript is well-written and comprehensively described. The references are appropriate and adequate.

Author Response

In this review, the authors illustrated DICER1's functions and how they relate to both cancer-causing and non-cancerous illnesses. DICER1, is a gene located on chromosome 14q32.13. It contains 27 exons linking the two enzyme domains. DICER1 is a vital housekeeping gene that encodes a multi-domain enzyme in small RNA processing. This enzyme functions in several pathways, including RNA interference, DNA damage repair, and antiviral responses. At the protein level, the endoribonuclease DICER is also involved in several human diseases, of which the pleuropulmonary blastoma is probably the most egregious entity.

Overall, this manuscript is well-written and comprehensively described. The references are appropriate and adequate.

Thank you for your comments. We revised the manuscript according to the comments we received and added two new sections, including limitations and future directions, as requested by other reviewers. We are deeply grateful to you and all reviewers.

 

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

This review on the pathological role Dicer/Argonaute endonuclease, to my knowledge on microRNA processing and pediatric, will interest the large reader community of Biology.

Author Response

This review on the pathological role Dicer/Argonaute endonuclease, to my knowledge on microRNA processing and pediatric, will interest the large reader community of Biology.

Thank you for your comments. We revised the manuscript according to the comments we received and added two new sections, including limitations and future directions.

We are deeply grateful to you and all reviewers.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have sufficiently revised the manuscript and it is now suitable for publication. 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, You've done a great job! I believe your review deserves to be published as soon as possible. I am also pleased with the additional detailed illustrations and diagrams.

The manuscript provides a comprehensive overview of the DICER1 gene and its significance.

The conclusion regarding the potential for nanotechnology-supported therapies and drug repurposing is intriguing.

Overall, the review presents important information about the DICER1 gene and its implications in human health. With some revisions for clarity, consistency, and specificity, it has the potential to be a valuable contribution to the literature

all the best

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is improved after revision. I recommend the acceptance of the manuscript