Long Noncoding RNA XIST Originating From Synovial Fluid Exosomes Are Involved In Female Osteoarthritis Via Synovial Cell Stimulation

BackgroundOsteoarthritis(OA)is a common chronic degenerative disease.Its prevalence is >30% in the elderly aged >65 years,and the aging population has contributed to the increasing incidence of OA.OA is characterized by the degeneration of articular cartilage,which manifests as pain,joint dysfunction,and deformity.This condition gradually develops toward deterioration,which severely affects the patient’s labor capacity and quality of life as well as causes a heavy economic burden on the patient’s family and society.The pathogenesis of OA is complex,and effective treatments to delay its progression are lacking.Interestingly,the incidence of OA shows a marked sex-specific difference,i.e.,its incidence is considerably higher in female than in male by two-to three-fold.Therefore,an in-depth discussion of sex-specific differential incidence is of critical significance for resolving the current OA-related cognitive and medical difficulties.Exosomes are tiny vesicle-like bodies secreted by cells,and they are widely involved in the transmission and communication of information across cells.They are secreted by different types of cells and carry different components,endowing them with various biological functions.Exosomes are key factors in the regulation of biological functions of various types of cells and tissues in many aspects of bone metabolism,bone development,and disease progression.Studies have found that all types of cells in human joints can secrete exosomes and thereby transmit pathological signals or promote inflammatory reactions to participate in cell-to-cell signal exchange and joint disease.OA synovial fluid exosomes have the function of transmitting regulatory signals across cells.Recently,the role of RNA contained in synovial fluid exosomes in the development of OA has received increasing attention.However,the expression pattern of long noncoding RNA(lnc RNA)in OA synovial fluid exosomes remains unclear,and its role and mechanism in the progression of OA require further study.ObjectiveWe sought to elucidate the differential expression patterns of lnc RNA in synovial fluid exosomes in OA patients of both sexes,to screen and study how the key differential lnc RNAs stimulate synovial cells to participate in the development of OA,and to explore possible internal mechanisms.MethodsSynovial fluid samples were collected from three different male and female OA patients during surgery,and OA synovial fluid exosomes were extracted by polymer precipitation.To observe the general characteristics of OA synovial fluid exosomes,we used transmission electron microscopy to view their morphological characteristics,nanoparticle tracking analysis(NTA)to observe their distribution,and western blotting to identify the specific marker molecules CD81 and CD63 in OA synovial fluid exosomes.After RNA extraction,high-throughput RNA sequencing was performed,and further statistical and professional analyses of sequencing results were performed to screen differentially expressed lnc RNA.Real-time polymerase chain reaction(RT-PCR)was performed to validate important differentially expressed lnc RNA based on high-throughput sequencing RNA findings,particularly that of lnc RNA XIST.In female OA patients,synovial cells were isolated by type II collagenase digestion,and CD68 and Vimentin were detected by immunofluorescence after subculture to identify synovial cells.Synovial cells were incubated with Di I-labeled synovial fluid exosomes to observe whether synovial cells endocytosed OA synovial fluid exosomes.Expression and distribution of lnc RNA XIST in synovial cells were detected by RNA-fluorescence in situ hybridization experiments.Using RT-PCR,we tested the effect of siRNA on the expressions of certain genes by performing lnc RNA XIST knockdown in synovial cells derived from female OA patients.We performed RT-PCR to detect the effect of siRNA on levels of matrix metalloproteinases(MMPs);tissue inhibitors of MMPs;and inflammatory factors such as interleukin(IL)-1β,IL-6,and tumor necrosis factor(TNF)-αand the expressions of disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS)-5,cyclooxygenase(COX)-2,and vascular endothelial growth factor(VEGF),which are important molecules involved in OA.IL-1β-stimulated synovial cells were used to establish an in vitro cell inflammation model,and the effects of lnc RNA XIST knockdown on MMPs were observed.To explore the internal molecular mechanism underlying MMP regulation by lnc RNA XIST,RT-PCR was performed to detect the expression of Glut1 in synovial cells during lnc RNA XIST knockdown.ResultsThe size of OA synovial fluid exosome was 100–120 nm,which is consistent with the size of a typical saucer-like exosome structure.NTA revealed that particle size distribution was mainly between 80 and 200 nm,and particle concentration was highest at 152 nm.CD63 and CD81 were significantly expressed.lnc RNA expression pattern was considerably different between male and female OA patients: 84 lnc RNAs were upregulated and 53 were downregulated.Screening for markedly different lnc RNAs revealed the change in the expression pattern of lnc RNA XIST to be the most obvious.Further verification by RT-PCR showed that four lnc RNAs(XIST,MEG3,CYTOR,and FTX)were considerably upregulated and three lnc RNAs(AC118758.3,NEAT1,and TTC28-AS1)were considerably downregulated.Synovial cells were isolated and subcultured.The cells showed a long spindle shape and partly formed a vortex structure.CD68(-)and Vimentin(+)were comparable with fibroblast-like synovial cells in terms of the latter’s typical characteristics.Incubation of synovial cells with Di I-labeled OA synovial fluid exosomes caused production of red fluorescence and increased the expression of lnc RNA XIST,the most significant difference in synovial fluid exosomes between male and female OA patients.Synovial cells were incubated with those of the opposite sex.Compared with those derived from male OA patients,the expression of lnc RNA XIST was higher in synovial cells derived from female OA patients.Synovial cells express lnc RNA XIST molecules in the nucleus and cytoplasm.siRNA significantly inhibited the expression of lnc RNA XIST in synovial cells,and this downregulation of lnc RNA XIST significantly inhibited the expressions of the OA-related MMPs MMP-3 and MMP-13.However,lnc RNA XIST knockdown had no significant effect on synovitis-related inflammatory factors(IL-1β,IL-6,and TNF-α)and important molecules involved in pathological changes in OA such as ADAMTS-5,COX-2,and VEGF.In the IL-1β-stimulated cell inflammation model,lnc RNA XIST knockdown significantly inhibited the expressions of MMP-1,MMP-3,and MMP-13 in synovial cells.lnc RNA XIST knockdown also regulated the expression of Glut1 in resting synovial cells in OA.Further experimental results showed that lnc RNA XIST knockdown significantly inhibited the expression of Glut1 in the IL-1β-stimulated inflammation model in vitro.ConclusionSynovial fluid is rich in exosomes.OA synovial fluid exosomes share typical characteristics with other body-fluid exosomes.lnc RNA XIST expression significantly increased in OA synovial fluid exosomes derived from female OA patients compared with in those derived from male OA patients.Synovial fluid exosomes were endocytosed by synovial cells,suggesting that synovial cells can be used as target cells for effective transmission of biological information.lnc RNA XIST stimulates synovial cells to regulate the expressions of MMP molecules and regulate OA synovitis.Thus,lnc RNA XIST may be involved in the occurrence and development of OA by regulating the expression of Glut1 in synovial cells.