Biological sample collection to advance research and treatment: a Fight Osteosarcoma Through European Research (FOSTER) and Euro Ewing Consortium (EEC) statement.

Osteosarcoma and Ewing sarcoma are bone tumours mostly diagnosed in children, adolescents and young adults. Despite multi-modal therapy, morbidity is high and survival rates remain low, especially in the metastatic disease setting. Trials investigating targeted therapies and immunotherapies have not been ground-breaking. Better understanding of biological subgroups, the role of the tumour immune microenvironment, factors that promote metastasis and clinical biomarkers of prognosis and drug response are required to make progress. A prerequisite to achieve desired success is a thorough, systematic and clinically linked biological analysis of patient samples but disease rarity and tissue processing challenges such as logistics and infrastructure have contributed to a lack of relevant samples for clinical care and research. There is a need for a Europe-wide framework to be implemented for the adequate and minimal sampling, processing, storage and analysis of patient samples. Two international panels of scientists, clinicians and patient and parent advocates have formed the Fight Osteosarcoma Through European Research (FOSTER) consortium and the Euro Ewing Consortium (EEC). The consortia shared their expertise and institutional practices to formulate new guidelines. We report new reference standards for adequate and minimally required sampling (time points, diagnostic samples, liquid biopsy tubes), handling and biobanking to enable advanced biological studies in bone sarcoma. We describe standards for analysis and annotation to drive collaboration and data harmonisation with practical, legal and ethical considerations. This position paper provides comprehensive guidelines that should become the new standards of care that will accelerate scientific progress, promote collaboration and improve outcomes.

Super-enhancers: drivers of cells' identities and cells' debacles.

Precise spatiotemporal regulations of gene expression are essential for determining cells' fates and functions. Enhancers are cis-acting DNA elements that act as periodic transcriptional thrusters and their activities are cell type specific. Clusters of enhancers, called super-enhancers, are more densely occupied by transcriptional activators than enhancers, driving stronger expression of their target genes, which have prominent roles in establishing and maintaining cellular identities. Here we review the current knowledge on the composition and structure of super-enhancers to understand how they robustly stimulate the expression of cellular identity genes. We also review their involvement in the development of various cell types and both noncancerous and cancerous disorders, implying the therapeutic interest of targeting them to fight against various diseases.

Ewing sarcoma from molecular biology to the clinic.

In Europe, with an incidence of 7.5 cases per million, Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children, adolescents and young adults, after osteosarcoma. Since the 1980s, conventional treatment has been based on the use of neoadjuvant and adjuvant chemotherapeutic agents combined with surgical resection of the tumor when possible. These treatments have increased the patient survival rate to 70% for localized forms, which drops drastically to less than 30% when patients are resistant to chemotherapy or when pulmonary metastases are present at diagnosis. However, the lack of improvement in these survival rates over the last decades points to the urgent need for new therapies. Genetically, ES is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family. In 85% of cases, the chromosomal translocation found is (11; 22) (q24; q12), between the EWS RNA-binding protein and the FLI1 transcription factor, leading to the EWS-FLI1 fusion protein. This chimeric protein acts as an oncogenic factor playing a crucial role in the development of ES. This review provides a non-exhaustive overview of ES from a clinical and biological point of view, describing its main clinical, cellular and molecular aspects.

Tracking Targets of Dynamic Super-Enhancers in Vitro to Better Characterize Osteoclastogenesis and to Evaluate the Effect of Diuron on the Maturation of Human Bone Cells.

BACKGROUND: Osteoclasts are major actors in the maintenance of bone homeostasis. The full functional maturation of osteoclasts from monocyte lineage cells is essential for the degradation of old/damaged bone matrix. Diuron is one of the most frequently encountered herbicides, particularly in water sources. However, despite a reported delayed ossification in vivo, its impact on bone cells remains largely unknown. OBJECTIVES: The objectives of this study were to first better characterize osteoclastogenesis by identifying genes that drive the differentiation of CD14+ monocyte progenitors into osteoclasts and to evaluate the toxicity of diuron on osteoblastic and osteoclastic differentiation in vitro. METHODS: We performed chromatin immunoprecipitation (ChIP) against H3K27ac followed by ChIP-sequencing (ChIP-Seq) and RNA-sequencing (RNA-Seq) at different stages of differentiation of CD14+ monocytes into active osteoclasts. Differentially activated super-enhancers and their potential target genes were identified. Then to evaluate the toxicity of diuron on osteoblasts and osteoclasts, we performed RNA-Seq and functional tests during in vitro osteoblastic and osteoclastic differentiation by exposing cells to different concentrations of diuron. RESULTS: The combinatorial study of the epigenetic and transcriptional remodeling taking place during differentiation has revealed a very dynamic epigenetic profile that supports the expression of genes vital for osteoclast differentiation and function. In total, we identified 122 genes induced by dynamic super-enhancers at late days. Our data suggest that high concentration of diuron (50µM) affects viability of mesenchymal stem cells (MSCs) in vitro associated with a decrease of bone mineralization. At a lower concentration (1µM), an inhibitory effect was observed in vitro on the number of osteoclasts derived from CD14+ monocytes without affecting cell viability. Among the diuron-affected genes, our analysis suggests a significant enrichment of genes targeted by pro-differentiation super-enhancers, with an odds ratio of 5.12 (ρ=2.59×10-5). DISCUSSION: Exposure to high concentrations of diuron decreased the viability of MSCs and could therefore affect osteoblastic differentiation and bone mineralization. This pesticide also disrupted osteoclasts maturation by impairing the expression of cell-identity determining genes. Indeed, at sublethal concentrations, differences in the expression of these key genes were mild during the course of in vitro osteoclast differentiation. Taken together our results suggest that high exposure levels of diuron could have an effect on bone homeostasis. https://doi.org/10.1289/EHP11690.

The p53 Family Members p63 and p73 Roles in the Metastatic Dissemination: Interactions with microRNAs and TGFß Pathway.

TP53 (TP53), p73 (TP73), and p63 (TP63) are members of the p53 transcription factor family, which has many activities spanning from embryonic development through to tumor suppression. The utilization of two promoters and alternative mRNA splicing has been shown to yield numerous isoforms in p53, p63, and p73. TAp73 is thought to mediate apoptosis as a result of nuclear accumulation following chemotherapy-induced DNA damage, according to a number of studies. Overexpression of the nuclear ΔNp63 and ΔNp73 isoforms, on the other hand, suppresses TAp73's pro-apoptotic activity in human malignancies, potentially leading to metastatic spread or inhibition. Another well-known pathway that has been associated to metastatic spread is the TGF pathway. TGFs are a family of structurally related polypeptide growth factors that regulate a variety of cellular functions including cell proliferation, lineage determination, differentiation, motility, adhesion, and cell death, making them significant players in development, homeostasis, and wound repair. Various studies have already identified several interactions between the p53 protein family and the TGFb pathway in the context of tumor growth and metastatic spread, beginning to shed light on this enigmatic intricacy.

Origin and Therapies of Osteosarcoma.

Osteosarcoma (OS) is the most frequent primary bone tumor, mainly affecting children and young adults. Despite therapeutic advances, the 5-year survival rate is 70% but drastically decreases to 20-30% for poor responders to therapies or for patients with metastasis. No real evolution of the survival rates has been observed for four decades, explained by poor knowledge of the origin, difficulties related to diagnosis and the lack of targeted therapies for this pediatric tumor. This review will describe a non-exhaustive overview of osteosarcoma disease from a clinical and biological point of view, describing the origin, diagnosis and therapies.

Involvement of the TGF-ß Signaling Pathway in the Development of YAP-Driven Osteosarcoma Lung Metastasis.

BACKGROUND: The poor survival rate of patients with osteosarcoma (OS), specifically with metastases at diagnosis, undergoes the urgency to develop new therapeutic strategies. Although we recently demonstrated the key role of YAP/TEAD signaling in the growth of OS primary tumor, the molecular mechanisms by which YAP regulates metastases development remain poorly understood. METHODS: The molecular mechanisms by which YAP regulates metastases development were studied using an overexpression of mutated forms of YAP able or not able to interact with TEAD. Molecular signatures were identified using RNA-sequencing analysis and gene set enrichment. Interactions between YAP and Smad3 were studied using proximity ligation assay (PLA), immunoprecipitation, and promoter/specific gene assays. The involvement of the TGF-ß pathway in the ability of YAP to stimulate metastatic development in vivo was studied using an inhibitor of the TGF-ß cascade in a preclinical model of OS and in vitro on the ability of OS cells to migrate and invade. RESULTS: Our work shows that a high YAP expression is associated with the presence of lung metastases which predicts a poor prognosis. Molecular analysis indicates that TGF-ß signaling is involved in YAP-driven osteosarcoma cell pro-migratory phenotype, epithelial mesenchymal transition, cell migration, and in vivo lung metastasis development. Regardless of its ability to bind to TEAD, YAP interacts with Smad3 and stimulates the transcriptional activity of TGF-ß/Smad3, thereby enhancing the ability of TGF-ß to stimulate lung metastasis development. CONCLUSIONS: We demonstrated the crucial involvement of the TGF-ß/Smad3 signaling pathway in YAP-driven lung metastasis development in OS.

Ubiquitin-specific proteases as therapeutic targets in paediatric primary bone tumours?

In children and young adults, primary malignant bone tumours are mainly composed of osteosarcoma and Ewing's sarcoma. Despite advances in treatments, nearly 40% of patients succumb to these diseases. In particular, the clinical outcome of metastatic osteosarcoma or Ewing's sarcoma remains poor, with less than 30% of patients who develop metastases surviving five years after initial diagnosis. Over the last decade, the cancer research community has shown considerable interest in the processes of protein ubiquitination and deubiquitination. In particular, a growing number of studies show the relevance to target the ubiquitin-specific protease (USP) family in various cancers. This review provides an update on the current knowledge regarding the implication of these USPs in the progression of bone sarcoma: osteosarcoma and Ewing's sarcoma.

Overexpression of the Ubiquitin Specific Proteases USP43, USP41, USP27x and USP6 in Osteosarcoma Cell Lines: Inhibition of Osteosarcoma Tumor Growth and Lung Metastasis Development by the USP Antagonist PR619.

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan-Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

Molecular Chaperones in Osteosarcoma: Diagnosis and Therapeutic Issues.

Osteosarcoma (OS) is the most common form of primary bone tumor affecting mainly children and young adults. Despite therapeutic progress, the 5-year survival rate is 70%, but it drops drastically to 30% for poor responders to therapies or for patients with metastases. Identifying new therapeutic targets is thus essential. Heat Shock Proteins (HSPs) are the main effectors of Heat Shock Response (HSR), the expression of which is induced by stressors. HSPs are a large family of proteins involved in the folding and maturation of other proteins in order to maintain proteostasis. HSP overexpression is observed in many cancers, including breast, prostate, colorectal, lung, and ovarian, as well as OS. In this article we reviewed the significant role played by HSPs in molecular mechanisms leading to OS development and progression. HSPs are directly involved in OS cell proliferation, apoptosis inhibition, migration, and drug resistance. We focused on HSP27, HSP60, HSP70 and HSP90 and summarized their potential clinical uses in OS as either biomarkers for diagnosis or therapeutic targets. Finally, based on different types of cancer, we consider the advantage of targeting heat shock factor 1 (HSF1), the major transcriptional regulator of HSPs in OS.

Mechanisms of Resistance to Conventional Therapies for Osteosarcoma.

Osteosarcoma (OS) is the most common primary bone tumor, mainly occurring in children and adolescents. Current standard therapy includes tumor resection associated with multidrug chemotherapy. However, patient survival has not evolved for the past decades. Since the 1970s, the 5-year survival rate is around 75% for patients with localized OS but dramatically drops to 20% for bad responders to chemotherapy or patients with metastases. Resistance is one of the biological processes at the origin of therapeutic failure. Therefore, it is necessary to better understand and decipher molecular mechanisms of resistance to conventional chemotherapy in order to develop new strategies and to adapt treatments for patients, thus improving the survival rate. This review will describe most of the molecular mechanisms involved in OS chemoresistance, such as a decrease in intracellular accumulation of drugs, inactivation of drugs, improved DNA repair, modulations of signaling pathways, resistance linked to autophagy, disruption in genes expression linked to the cell cycle, or even implication of the micro-environment. We will also give an overview of potential therapeutic strategies to circumvent resistance development.

Development of prohibitin ligands against osteoporosis.

Current therapeutic approaches to osteoporosis display some potential adverse effects and a limited efficacy on non-vertebral fracture reduction. Some sulfonylamidines targeting the scaffold proteins prohibitins-1 and 2 (PHB1/2) have been showed to inhibit the formation of osteoclasts in charge of bone resorption. Herein, we report the development of a second generation of anti-osteoclastic PHB ligands. The most potent compound, IN45, showed 88% inhibition at the low concentration of 5 µM, indicates that it might serve as a basis for the development of new antiosteoporotic drugs.

Sonic Hedgehog Signature in Pediatric Primary Bone Tumors: Effects of the GLI Antagonist GANT61 on Ewing's Sarcoma Tumor Growth.

Osteosarcoma (OS) and Ewing's sarcoma (ES) are the most common malignant bone tumors in children and adolescents. In many cases, the prognosis remains very poor. The Sonic hedgehog (SHH) signaling pathway, strongly involved in the development of many cancers, regulate transcription via the transcriptional factors Gli1-3. In this context, RNAseq analysis of OS and ES cell lines reveals an increase of some major compounds of the SHH signaling cascade in ES cells, such as the transcriptional factor Gli1. This increase leads to an augmentation of the transcriptional response of Gli1 in ES cell lines, demonstrating a dysregulation of Gli1 signaling in ES cells and thus the rationale for targeting Gli1 in ES. The use of a preclinical model of ES demonstrates that GANT61, an inhibitor of the transcriptional factor Gli1, reduces ES primary tumor growth. In vitro experiments show that GANT61 decreases the viability of ES cell, mainly through its ability to induce caspase-3/7-dependent cell apoptosis. Taken together, these results demonstrates that GANT61 may be a promising therapeutic strategy for inhibiting the progression of primary ES tumors.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

N6-Adenosine Methylation of miRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool.

MicroRNAs (miRNAs or miRs) play crucial roles in biological and pathological processes. Some miRNAs also appear as promising biomarkers and therapeutic tools. However, the epitranscriptomic regulation of miRNAs is not yet fully elucidated in all of their fields of application. We report that adenosine methylation of miR-200b-3p inhibits its repressive function toward its mRNA targets such as XIAP by blocking the formation of the miRNA/3' UTRmRNA duplex. Our data indicate that the adenosine methylation of miR-200b-3p is associated with the survival of glioblastoma patients. Collectively, our data support the idea that the adenosine methylation of miR-200b-3p can be used as a prodrug having a selective cytotoxicity against cancer cells (while being harmless to peripheral blood mononuclear cells [PBMCs], astrocytes, neurons, and hepatocytes).

Ribosomopathies: New Therapeutic Perspectives.

Ribosomopathies are a group of rare diseases in which genetic mutations cause defects in either ribosome biogenesis or function, given specific phenotypes. Ribosomal proteins, and multiple other factors that are necessary for ribosome biogenesis (rRNA processing, assembly of subunits, export to cytoplasm), can be affected in ribosomopathies. Despite the need for ribosomes in all cell types, these diseases result mainly in tissue-specific impairments. Depending on the type of ribosomopathy and its pathogenicity, there are many potential therapeutic targets. The present manuscript will review our knowledge of ribosomopathies, discuss current treatments, and introduce the new therapeutic perspectives based on recent research. Diamond-Blackfan anemia, currently treated with blood transfusion prior to steroids, could be managed with a range of new compounds, acting mainly on anemia, such as L-leucine. Treacher Collins syndrome could be managed by various treatments, but it has recently been shown that proteasomal inhibition by MG132 or Bortezomib may improve cranial skeleton malformations. Developmental defects resulting from ribosomopathies could be also treated pharmacologically after birth. It might thus be possible to treat certain ribosomopathies without using multiple treatments such as surgery and transplants. Ribosomopathies remain an open field in the search for new therapeutic approaches based on our recent understanding of the role of ribosomes and progress in gene therapy for curing genetic disorders.

MicroRNA-17-5p Reduces Inflammation and Bone Erosions in Mice With Collagen-Induced Arthritis and Directly Targets the JAK/STAT Pathway in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

OBJECTIVE: We undertook this study to examine microRNA (miRNA) expression across rheumatoid arthritis (RA) phenotypes, along with the effects and mechanisms of action of miRNA-17-5p (miR-17). METHODS: A miRNA array was performed in synovial tissue biopsied from patients with naive erosive RA (n = 3) and patients with nonerosive RA (n = 3). MicroRNA-17 lipoplex was delivered intraarticularly in the murine collagen-induced arthritis model. Clinical, histologic, and structural effects were studied over the course of arthritis. In-depth studies of the mechanisms of action of miR-17 were performed in primary RA fibroblast-like synoviocytes (FLS) isolated from synovial tissue. RESULTS: Fifty-five miRNAs including miR-17 were reduced in erosive RA. The miR-17 transfection into arthritic paws reduced the clinical inflammation score between day 2 and day 7 (2.8 versus 1.9; P = 0.03). Synovial B cell, T cell, macrophage, and polynuclear neutrophil infiltration was significantly reduced. Structural damage was also decreased, as shown by a reduction in the number of osteoclasts detected using tartrate-resistant acid phosphatase staining (osteoclast surface/bone surface 32% versus 18%; P = 0.005) and erosion score by computed tomography analysis (2.9 versus 1.7; P = 0.023). Proinflammatory cytokines from the interleukin-6 (IL-6) family and IL-1ß expression were also significantly reduced, but tumor necrosis factor was not. MicroRNA-17 directly targeted the 3'-untranslated regions of STAT3 and JAK1. STAT3 and JAK1 messenger RNA (mRNA) and protein expression were reduced in RA FLS following miR-17 transfection. STAT3 and JAK1 mRNA and activation of STAT3, as assessed by immunohistochemistry, were also reduced in injected paws (% stained area 93% versus 62%; P = 0.035). CONCLUSION: We demonstrate an antiinflammatory and antierosive role of miR-17 in vivo. This effect involves the suppression of the IL-6 family autocrine-amplifying loop through the direct targeting of JAK1 and STAT3.

Implication of the p53-Related miR-34c, -125b, and -203 in the Osteoblastic Differentiation and the Malignant Transformation of Bone Sarcomas.

The formation of the skeleton occurs throughout the lives of vertebrates and is achieved through the balanced activities of two kinds of specialized bone cells: the bone-forming osteoblasts and the bone-resorbing osteoclasts. Impairment in the remodeling processes dramatically hampers the proper healing of fractures and can also result in malignant bone diseases such as osteosarcoma. MicroRNAs (miRNAs) are a class of small non-coding single-strand RNAs implicated in the control of various cellular activities such as proliferation, differentiation, and apoptosis. Their post-transcriptional regulatory role confers on them inhibitory functions toward specific target mRNAs. As miRNAs are involved in the differentiation program of precursor cells, it is now well established that this class of molecules also influences bone formation by affecting osteoblastic differentiation and the fate of osteoblasts. In response to various cell signals, the tumor-suppressor protein p53 activates a huge range of genes, whose miRNAs promote genomic-integrity maintenance, cell-cycle arrest, cell senescence, and apoptosis. Here, we review the role of three p53-related miRNAs, miR-34c, -125b, and -203, in the bone-remodeling context and, in particular, in osteoblastic differentiation. The second aim of this study is to deal with the potential implication of these miRNAs in osteosarcoma development and progression.

TH1579, MTH1 inhibitor, delays tumour growth and inhibits metastases development in osteosarcoma model.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers. METHODS: The aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases. FINDINGS: MTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90 mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases. INTERPRETATION: All these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma. FUNDING: This study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé.

Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.

BACKGROUND: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.