RIOK3 potentially regulates osteogenesis related pathways in ankylosing spondylitis and the differentiation of bone marrow mesenchymal stem cells

Background: RNA-binding proteins (RBPs) are key effectors of gene expression andplay critical rolesininflammation, immune regulation,andmany rheumatic diseases. This study screened the differentially expressed RBPs between ankylosing spondylitis (AS) patients and the healthy control group through transcriptome sequencing and explored the potential biological function and mechanism of target RBP in AS patients. Methods: The differentially expressed genes (DEGs) in whole blood cells of 5 AS patients and 3 healthy people were obtained by RNA-seq, and the differentially expressed RBPs were obtained by overlapping the DEGs and the summarytable of RBPs. The selected target RBP (RIOK3) was verified by qRT-PCR in the peripheral blood mononuclear cells (PBMCs) of patients with AS and age_x001E_ and sex_x001E_ matched controls. Next, mouse bone marrow mesenchymal stem cells (mBMSCs) were transfected with siRNA targeting RIOK3 usingthelipofectaminetransfectiontechnique, and the molecular functions, biological processes, and signaling pathway enrichment analysis of DEGs regulated by RIOK3 were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The expression level of the selected genes in siRIOK3 mBMSCs was detected by qRT-PCR to further verify the regulatory role of RIOK3. Then, the RIOK3 stable knockdown transfected cell lines were constructed by lentivirus infection, and calcium deposits were observed byAlizarinredstaining after21daysof osteogenicinduction, and the expression of osteogenic genes ALP, OCN, and OPN were assessed by qRT-PCR. Results: RIOK3 was enriched in interferon-related pathway and innate immune-related pathway, which was an important immune regulatory factor and could effected the function of ribosomes. RIOK3knockdown will inhibited the expression of genes related to transcription and ribosomal function in mBMSCs, especially the transcription factors related to osteogenic differentiation, and will also effected the expression of β-interferon related pathway genes. RIOK3 knockdown effect was mainly co-expressed with HIF-1 signal pathway, MAPK signal pathway, and inflammatory response related pathway genes in the osteogenic differentiation of mBMSCs. The cellularexperimentalresults further confirmedthat RIOK3 knockdown will reduce the osteogenic differentiation ability of mBMSCs. Conclusion: Collectively,RIOK3 may affect the differentiation of mBMSCs and participate in the occurrence and development of AS, especially pathological bone formation. Thisstudymay providenew perspectivefortheresearchofthe mechanism of AS and atheoreticalbasisforRIOK3asapotential target for AS therapy.