RIOK3 Potentially Regulates Osteogenesis Related Pathways In Ankylosing Spondylitis And The Differentiation Of Bone Marrow Mesenchymal Stem Cells

ObjectiveAnkylosing spondylitis(AS)is an immune-mediated chronic inflammatory rheumatic disease,which can cause inflammatory back pain,and affect the spine and sacroiliac joints,resulting in a decreased quality of life for patients and imposing a considerable burden on patients and society.AS is characterized by inflammation and pathologic new bone formation.In the past 10 years,the progress of high-throughput technology and cohort studies involving large samples have made considerable progress in the new genetic association and immune pathway of AS.The discovery of several inflammatory pathways has put AS into the era of biological agents,providing new options for the treatment of AS.Despite these advances,the underlying pathogenesis of the disease has not been fully understood.There are large individual differences between different patients in response to the existing treatment.Therefore,exploring the pathogenesis of AS may help us find new specific biomarkers that affect the progress of AS,especially the formation of new bone,which may provide new ideas for the evaluation or treatment of AS.RNA-binding proteins(RBPs)are key effectors of gene expression,play a regulatory role in different biological processes of various RNA,which have important biological functions in inflammation and immune regulation,and play a crucial role in the development and progression of various rheumatic diseases.This study screened the differentially expressed RBP between AS patients and the healthy control group through transcriptome sequencing and discussed the expression,clinical value,potential biological function,and mechanism of target RBP in AS patients.In this study,we screened the differentially expressed RBPs between AS patients and healthy control group through transcriptome sequencing,and investigate the differential expression,clinical values,potential biological function,and mechanism of target RBP in AS.Methods1.The differentially expressed genes in whole blood cells of 5 AS patients and 3 healthy people were obtained by RNA-seq,and the differentially expressed RBPs was obtained by overlapping the differentially expressed genes and the summary table of RBPs.Differentially expressed RBPs that are consistently expressed in patients and significantly differ from healthy adults,with GO and KEGG enrichment in inflammatory or osteogenesis-related pathways,were selected as target RBPs.2.qRT-PCR was used to validate the differential expression of RIO kinase 3(RIOK3)in AS patients and normal control group.We collected the general clinical data,evaluated the disease function index,disease activity index and imaging index of all enrolled patients,and compared the difference of RIOK3 expression in different disease activity states and degrees of structural damage.Linear regression model were used to investigate the factors influencing the measured data.Single factor and multiple factor logistic regression models were used to investigate the influencing factors of the count data,including single factor and multiple factor logistic regression.3.RIOK3 in mouse bone marrow mesenchymal stem cells(mBMSCs)was knocked down by siRNA,and the transcriptome study of siRIOK3 mBMSCs was carried out by RNA-seq technology,including gene differential expression analysis,functional annotation,GO and KEGG analysis,to obtain the downstream differential expression genes affected by RIOK3.4.According to the obtained RNA-seq data of siRIOK3 mBMSCs,the genes with significant differences in transcription level and closely related to the osteogenic differentiation pathway of AS were selected,and the expression level of the selected genes in siRIOK3 mBMSCs was detected by qRT-PCR to further verify the regulatory role of RIOK3 on the genes related to the osteogenic differentiation pathway of AS.5.Download the transcriptome data set GSE113844 from the GEO database,mainly including the transcriptome data of mBMSCs before and three days after the induction of osteogenic differentiation.Integrate the transcriptome data with the si-RIOK3 data in mBMSCs to further explore the effect of RIOK3 on the differentiation of mBMSCs.6.The RIOK3 stable knockdown transfected cell lines were constructed by lentivirus infection.We carried out the CCK-8 assay to detect the effect of knockdown RIOK3 on cell proliferation.To study the effect of knockdown RIOK3 on osteogenesis of mBMSCs,calcium deposits were observed by Alizarin red staining after 21 days of osteogenic induction,and the expression of osteogenic genes ALP,OCN and OPN were assessed by qRT-PCR at 0,7,and 14 days.Results1.A total of 1103 differentially expressed genes,including 676 up-regulated genes and427 down-regulated genes,were obtained from AS patients and normal control group by high-throughput sequencing technology.After extracting the intersection of differentially expressed gene sets and summary table of 1542 RBPs,20 differentially expressed RBPs were determined.RIOK3 was selected as the target RBP based on the results of gene differential expression,enrichment analysis and literature research.2.qRT-PCR validation and clinical analysis results:(1)The expression of RIOK3 in patients with AS was significantly higher than that in the normal control group(P<0.05);In different structural injury states,the expression level of bone marrow edema in sacroiliac joint MRI and bone bridge in spinal column was higher in AS patients(P<0.05).(2)Spearman correlation analysis revealed that there was a correlation between RIOK3 level and disease activity index(CRP,ASDAScrp)and structural injury index(bone bridge formation,SPARCC,m SASSS)(P<0.05).(3)Regression analysis:RIOK3,age,ESR,and X-ray grade were included in the linear regression analysis model of m SASSS score.RIOK3(β=0.363,P=0.018)was positively correlated with m SASSS score.RIOK3,gender,sleeper wall distance,BASFI,and m SASSS were included in the linear regression analysis model of SPARCC.RIOK3(β=0.440,P=0.003)was positively correlated with SPARCC score.Single factor and multiple factor Logistic regression showed that RIOK3,age and X-ray grade of sacroiliac joint were the risk factors for bone bridge formation(P<0.05).Based on the results of logistic regression analysis,a nomograph prediction model for bone bridge formation in AS patients was established.The C index of this model was 0.909(95% CI 0.820-0.999),the fitting degree of the calibration curve was high,and the P value was greater than 0.05,with high accuracy and good fitting degree.3.The knockdown of RIOK3 in mBMSCs will inhibit the expression of genes related to transcription and ribosomal function,especially transcription factors related to osteogenic differentiation,and also affect the expression of β-interferon related pathway genes,as well as the expression of IL-17 pathway and TNF-a pathway genes closely related to AS inflammatory response and pathological bone formation.qRT-PCR detection showed that osteogenic differentiation related transcription factors HMGA2,HIF1 A,JUN,SOX4,interferon pathway genes IFIT1,IFI202 b,IFI204,IFI203,IL-17 and TNF-α were significantly down-regulated in siRIOK3 mBMSCs(P<0.001).4.Integrating the transcriptome data of mBMSC before and after osteogenic differentiation induction with the data in si-RIOK3 mBMSC,the results showed that RIOK3 knockdown effect is mainly co-expressed with inflammatory response related pathway genes in osteogenic differentiation.KEGG is mainly concentrated in HIF-1signal pathway and MAPK signal pathway.5.The knockdown of RIOK3 inhibits proliferation of mBMSC.Alizarin Red calcium staining showed no calcium nodule formation in the knockdown RIOK3(KDRIOK3)group at day 21 of osteogenic induction.On both days 7 and 14 after induction for osteogenic differentiation,the mRNA relative expression of osteogenic marker genes ALP and OPN in KD-RIOK3 group were lower compared to NC group respectively.There was no obvious difference in the level of OCN expression between the two groups on day 7,while the level of OCN expression in KD-RIOK3 group was significantly lower than the NC group on day 14(P<0.05).Conclusion1.RIOK3 is enriched in interferon-related pathway and innate immune-related pathway,which is an important immune regulatory factor and can affect the function of ribosomes.2.RIOK3 is up-regulated in AS patients and related to AS disease activity and structural damage indicators.It is one of the risk factors of bone bridge development and may be an effective predictor of bone bridge development in AS patients.3.Knockdown RIOK3 will inhibit the expression of genes related to transcription and ribosomal function in mBMSCs,especially the transcription factors related to osteogenic differentiation,and will also affect the expression of β-interferon related pathway genes,as well as the expression of IL-17 pathway and TNF-a pathway genes closely related to AS inflammatory response and pathological bone formation.4.RIOK3 knockdown effect is mainly co-expressed with HIF-1 signal pathway,MAPK signal pathway,and inflammatory response related pathway genes in the osteogenic differentiation of mBMSCs5.The knockdown of RIOK3 inhibits the proliferation of mBMSC,reduces the expression levels of osteogenic markers such as ALP,OCN,and OPN,and inhibits osteogenic differentiation of BMSCs.