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IGF2BP3-mediated m6A modification of RASGRF1 promoting joint injury in rheumatoid arthritis [RIP-seq]

ABSTRACT: With the deepening of epigenetic research, more and more studies have shown that N6- methyladenosine (m6A) is closely related to the occurrence and development of rheumatoid arthritis (RA), but the specific mechanism still needs to be further elucidated. In the study, we collected synovial tissues from normal controls and patients with OA or RA. The levels of m6A and inflammation were analyzed by immunofluorescence and western blot. The role of IGF2BP3 in cell proliferation and inflammatory activation was explored using transfection and RNA immunoprecipitation assays. IGF2BP3-/- mice were established, which were used to construct a mouse model of arthritis by transferring serum from adult arthritis K/BxN mice. Histological analysis and micro-CT were performed to determine joint damage. m6A level was markedly increased in RA patients and mouse models, and the expression of IGF2BP3 was upregulated in RA and related to inflammatory markers. IGF2BP3 played an important part in RA-fibroblast-like synoviocytes (FLS) by promoting cell proliferation, migration, invasion, inflammatory cytokine release, and inhibiting autophagy. In addition, IGF2BP3 inhibited autophagy to reduce the production of ROS, thereby decreasing inflammatory activation in macrophages. More importantly, RASGRF1-mediated mTORC1 activation played a crucial role in the ability of IGF2BP3 to promote cell proliferation and inflammatory activation. Based on the arthritis model of IGF2BP3-/- mice, IGF2BP3 knockdown Inhibited RA-FLS proliferation and inflammatory infiltration, and further improved RA joint injury. Our study revealed the important role of IGF2BP3 in RA progression. The targeted inhibition of IGF2BP3 reduced cell proliferation and inflammatory activation and limited RA development in a mouse model of arthritis, providing a potential strategy for RA therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE273246 | GEO | 2025/05/28

REPOSITORIES: GEO

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Project description:The variant rs26232, in the first intron of the C5orf30 locus, has recently been associated with both risk of developing rheumatoid arthritis (RA) and severity of tissue damage. The biological activities of human C5orf30 are unknown, and neither the gene nor protein show significant homology to any other characterized human sequences. The C5orf30 gene is present only in vertebrate genomes with a high degree of conservation implying a central function in these organisms. Here we report that C5orf30 is highly expressed in the synovium of RA patients compared with control synovial tissue, and that it is predominately expressed by synovial fibroblast (RASF) and macrophages in the lining and sublining layer of the tissue. These cells play a central role in the initiation and perpetuation of RA and are implicated in cartilage destruction. RASFs lacking C5orf30 exhibit increased cell migration and invasion in vitro and gene-profiling following C5orf30 inhibition confirmed upregulation of genes involved in cell migration, adhesion, angiogenesis, and immune and inflammatory pathways. Importantly, loss of C5orf30 contributes to the pathology of inflammatory arthritis in vivo, since inhibition of C5orf30 in the collagen-induced arthritis model markedly accentuated joint inflammation and tissue damage. Our study reveal C5orf30 to be a novel negative regulator of tissue damage in RA and this may act by modulating the autoaggressive phenotype that is characteristic of RASFs. Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease that affects synovial joints. A key characteristic of RA is hyperplasia of fibroblast-like synoviocytes (FLS) which develop a stable, auto-aggressive phenotype that augments tissue destruction. It is unknown how this phenotype is stably maintained; however, epigenetic changes have been implicated. Histone deacetylation is one proposed method; a process controlled by histone deacetylases (HDACs). However, there have recently been reports publishing conflicting data regarding the expression of HDACs in RA synovium and FLS. The objective of this thesis is to determine the role of HDACs in regulating the auto-aggressive phenotype of RA through studies in FLS and in mice. Real time-quantitative PCR was used to assess the levels of HDAC1-11 in RA compared to osteoarthritis (OA) FLS. Immunohistochemistry and western blotting were used to assess protein expression of HDAC1 in RA and OA synovial tissue and FLS. HDAC1 was found to be overexpressed in RA compared to OA. HDAC1 was knocked down in RA FLS, then cell proliferation, migration and invasion were assessed by using tritiated thymidine, a scratch assay and a Matrigel invasion assay respectively. All three functions were significantly reduced following HDAC1 knockdown. An Illumina BeadChip (47,000 transcripts) was used to analyse global gene expression changes after knockdown. This revealed significant gene changes in important functional clusters, such as proliferation and migration. HDAC1 knockout is embryonic lethal in mice, so the in vivo role of HDAC1 was investigated in a mouse model of collagen-induced arthritis (CIA) using in vivo siRNAs. Clinical scores of CIA were measured daily and HDAC1 knockdown mice showed a significantly reduced clinical score compared to controls, comparable to dexamethasone-treated mice. The bones were analysed using a microCT scanner and histology. Knocking down HDAC1 showed reduced bone erosion, joint inflammation and cartilage degradation compared to controls. Overall, this study shows that HDAC1 is dysregulated in RA and it has a significant role in the autoaggressive phenotype shown in RA FLS and collagen-induced arthritis. The novel data shown in this thesis demonstrates that inhibiting HDAC1 may provide a powerful new target for treating RA. Three independent patient RASF samples were analysed in this study for each siRNA gene knockdown. Also a non targerting control siRNA was also used for each of the patient RASFs

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IGF2BP3-mediated m6A modification of RASGRF1 promoting joint injury in rheumatoid arthritis [RIP-seq]

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