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Project description:FTO-mediated RNA m6A demethylation regulates aggressive behavior and inflammation of fibroblast-like synoviocytes
| PRJNA1007578 | ENA
Project description:Polymorphism of the FTO gene encoding an N(6)-methyladenosine (m(6)A) RNA demethylase was robustly associated with human obesity; however, the mechanism by which FTO affects metabolism, considering its emerging role in RNA modification, is still poorly understood. A new study published in Cell Research reports novel functions implicating FTO in the regulation of mRNA alternative splicing in the control of adipogenesis.
| S-EPMC4650590 | biostudies-other
Project description:ObjectivesIncreasing evidence have demonstrated the N6-methyladenosine (m6A) plays critical roles in osteoarthritis (OA) progression, but the role of m6A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m6A demethylase fat mass and obesity-associated protein (FTO) in OA progression.Materials and methodsThe FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2.ResultsFTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2, thereby alleviating OA cartilage damage.ConclusionsOur results attested that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.
| S-EPMC10067311 | biostudies-literature
Project description:Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m(6)A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m(6)A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m(6)A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.
| S-EPMC4288171 | biostudies-literature
Project description:N 6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA and associated with numerous cellular processes in health and disease. Up- and down-regulation of its "writer" or "eraser" proteins alter the global m6A level; however, modifying distinct m6A sites has remained elusive. We genetically fused the dioxygenase FTO responsible for m6A demethylation to RCas9 as an RNA-targeting module. The resulting RCas9-FTO retained demethylation activity and bound to RNA in a sequence-specific manner depending on the sgRNA and PAMmer. Using SCARLET analysis, we quantified the m6A level at a specific site and analyzed the effect of the PAM-to-m6A distance on activity. Sequence-specific demethylation by RCas9-FTO was tested on different RNA combinations and showed up to 15-fold sequence preference for target RNA compared to off-target RNA. Taken together, RCas9-FTO represents a new tool for sequence-specific demethylation of m6A in RNA that can be readily adapted to any given RNA sequence and opens the door to studying the function of distinct m6A sites.
| S-EPMC6800472 | biostudies-literature
Project description:N6-methyladenosine (m6A) acts as the most common mRNA modification in mammal cells. Fat Mass and Obesity-associated protein (FTO) is the firstly identified demethylase in the m6A modification. This research tries to discover the potential roles of FTO in the m6A modification in the hepatocellular carcinoma (HCC). FTO level was found to be up-regulated in the HCC tissue and cells. The over-expression of FTO was correlated to the poor prognosis of HCC individuals. Knockdown of FTO suppressed the proliferation and in vivo tumor growth, and induced the G0/G1 phase arrest. Mechanically, FTO triggered the demethylation of PKM2 mRNA and accelerated the translated production. Overall, this finding suggests the critical function of FTO in the HCC oncogenesis and its m6A modification.
| S-EPMC6789218 | biostudies-literature
Project description:Fibroblast-like synoviocytes (FLSs), play a key role in perpetuating synovial inflammation and bone erosion in rheumatoid arthritis (RA), however, the underlying mechanism(s) of RA FLSs activation and aggression remain unclear. Identifying endogenous proteins that selectively target FLSs is urgently needed. Here, we systematically identified that secreted modular calcium-binding protein 2 (SMOC2), was significantly increased in RA FLSs and synovial tissues. SMOC2 knockdown specifically regulated cytoskeleton remodeling and decreased the migration and invasion of RA FLSs. Mechanistically, cytoskeleton-related genes were significantly downregulated in RA FLSs with reduced SMOC2 expression, especially the motor protein myosin1c (MYO1C). SMOC2 controlled MYO1C expression by SRY-related high-mobility group box 4 (SOX4) and AlkB homolog 5 (ALKHB5) mediated-m6A modification through transcriptional and post-transcriptional regulation. Furthermore, intra-articular Ad-shRNA-SMOC2 treatment attenuated synovial inflammation as well as bone and cartilage erosion in rats with collagen-induced arthritis (CIA). Our findings suggest that increased SMOC2 expression in FLSs may contribute to synovial aggression and joint destruction in RA. SMOC2 may serve as a potential target against RA. SMOC2-mediated regulation of the synovial migration and invasion in RA FLSs. In RA FLSs, SMOC2 is significantly increased, leading to the increased level of MYO1C via SOX4-mediated transcriptional regulation and ALKBH5-mediated m6A modification, thereby causing cytoskeleton remodeling and promoting RA FLSs migration and invasion. The Figure was drawn by Figdraw.
| S-EPMC9747908 | biostudies-literature
Project description:ObjectiveFibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs.MethodsRA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined.ResultsCaspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS.ConclusionBlocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.
| S-EPMC8992463 | biostudies-literature
Project description:Rheumatoid arthritis (RA) is a common systemic autoimmune disorder of unknown etiology, which threatens public health. The regulatory role of tripartite motif‑containing 22 (TRIM22) has been reported in multiple types of cancers and disease, but not in RA. The aim of the present study was therefore to elucidate the potential roles and underlying mechanisms of TRIM22 in fibroblast‑like synoviocytes (FLSs) in RA. The Gene Expression Omnibus database was used to examine TRIM22 mRNA expression levels in synovial tissue samples of patients with RA and healthy controls. TRIM22 and forkhead box C1 (FOXC1) mRNA and protein expression levels in normal FLSs and RA‑FLSs were assessed using reverse transcription‑quantitative PCR (RT‑qPCR) and western blotting, respectively. The Cell Counting Kit‑8 assay was used to assess cell proliferation. Cell apoptosis was analyzed using flow cytometry. The migratory and invasive abilities of RA‑FLSs were assessed using Transwell assays. Western blotting was used to analyze the protein expression levels of apoptosis‑related factors, MMP2, MMP9 and NF‑κB signaling pathway‑related proteins. Inflammatory factors levels were assessed via ELISA and RT‑qPCR. Furthermore, the JASPAR database, chromatin immunoprecipitation and the dual‑luciferase reporter assays were used to determine the interaction between FOXC1 and the TRIM22 promoter. The results of the present study demonstrated that TRIM22 expression levels were significantly elevated in the synovial tissue samples of patients with RA and RA‑FLSs. Moreover, FOXC1 was also significantly overexpressed in RA‑FLSs. TRIM22 knockdown significantly reduced cell proliferation, migration, invasion and the inflammatory response, whereas cell apoptosis was significantly increased. Furthermore, the results demonstrated that FOXC1 may have positively mediated TRIM22 expression via binding to the TRIM22 promoter. Moreover, FOXC1 overexpression significantly reversed the outcome of TRIM22 knockdown on the proliferation, apoptosis, migration, invasion and inflammation of RA‑FLSs. FOXC1 overexpression also significantly reversed the inactivation of the NF‑κB signaling pathway caused by TRIM22 knockdown. In summary, the present study demonstrated that TRIM22 was potentially activated via FOXC1, which contributed to the progression of RA via the NF‑κB signaling pathway.
| S-EPMC9434987 | biostudies-literature
Project description:Fibroblast-like synoviocytes (FLS) are crucial in promoting articular inflammation and destruction in rheumatoid arthritis (RA). As the most abundant RNA modification, the function of m6A in RA FLS is still unclear. Here, we constructed FTO-knockdown FLS to explore the mechanism of FTO in regulating the aggressive behavior of RA FLS.
2024-12-31 | GSE241249 | GEO