| Backgroud and objective:Osteomyelitis is an acute or chronic bone tissue infection,characterized by suppurative inflammation,abnormal bone remodeling,and refractory bone resorption,often accompanied by bone defects,bone nonunion and other orthopedic diseases.Osteomyelitis often lead to limb dysfunction,amputation,and even life-threatening.Osteomyelitis is a serious threat to the physical and mental health of patients,and its treatment has been one of the problems faced by orthopedic surgeons.However,the mechanism of the development of osteomyelitis plus bone nonunion is not clear.Staphylococcus aureus is the most common microorganism that causes osteomyelitis.One of the key virulence factor of Staphylococcus aureus is staphylococcal protein A(SPA).Previous studies show that SPA can induce an inflammatory response.SPA can directly stimulate osteoblast apoptosis,leading to bone destruction and bone loss in lesion area,which indicated that SPA plays a key role in the development of osteomyelitis.In the process of bone regeneration,a sufficient number of BMSCs were gathered to the fracture site.The differentiation ability of BMSCs to osteoblasts directly affects the new bone formation and bone healing ability of bone tissue.Previous studies have focused on the SA and SPA induced the apoptosis of osteoblasts.The effects of SPA on the differentiation of BMSCs have not been reported.The effect of SPA on the osteogenic differentiation of BMSCs was firstly studied by our research osteogenic differentiation of BMSCs,suggesting that the virulence factor-SPA could simuteam in the early stage of this study.It was found that SPA could significantly inhibit the late the inflammatory environment in vivo.Meanwhile,how to affect SPA BMSCs osteogenesis differentiation mechanism is not very clear.So,it is important to study the causes of osteoblastic differentiation ability of hBMSCs under SPA sitmulation,which is of great significance for further understanding the causes of osteogenesis obstacles in osteomyelitis.Long noncoding RNAs(IncRNAs),non-protein coding transcripts longer than 200 nucleotides,have been implicated in a range of developmental processes and diseases.Previous studies indicate that some lncRNAs play a role in regulating osteogenic differentiation of stem cells,such as AK141205,ANCR,and MEG3.The above studies have shown that IncRNA has a positive regulatory effect on stem cell differentiation.And some studies have shown that some IncRNA stem cells on the osteogenic differentiation of negative regulation.It was found that overexpression of AK035085 inhibited the osteogenic differentiation of C3H10T1/2 cells.However,the complete expression profile and function of lncRNAs during the osteogenic differentiation of stem cells in an inflammatory microenvironment were not known.In the present study,hBMSCs were treated with different concentrations SPA to build osteomyelitis cell model by detected the expression of inflammatory cytokines and osteogenetic activity.Thus,this cell model was used to simulate osteomyelitis in hBMSCs in vitro.We then analyzed the whole expression profile of IncRNAs in SPA-treated hBMSCs using a lncRNA microarray.Further analysis that the effect of IncRNA by regulating its target gene on the osteogenic differentiation of hBMSCs induced by osteogenesis.And analyzed the mechanism that lncRNA regulate the hBMSCs osteogenic differentiation in osteomyelitis state.This study is the first time to explain the deficiency of osteogenesis ability of hBMSCs in osteomyelitis from IncRNA level,which provide a new theoretical and research direction for the study of osteomyelitis plus defect,and provide a solid foundation for the next research.Methods:1.The establishment of the SPA-induced osteomyelitis model in vitroFor construction of the osteomyelitis cell model,different concentrations of SPA(0?g/ml?0.1 ?g/ml,0.5 ?g/ml,1 ?g/ml,10 ?g/mL,100 ?g/mL)were added to treat hBMSCs.After treatment for 72 h,cell supernatants from the 24-well plates were harvested for IL-la,IL-6 and TNF-a by ELISA.For alizarin red staining,hBMSCs were treated with different concentrations of SPA and cultured for 3 weeks to detect the calcium nodules depositionand alkaline phosphatase(ALP)detection.2.The differential lncRNA were analyzed and verification in the process of SPA-induced hBMSC sosteogenic differentiationFor lncRNA microarray analysis,hBMSCs were cultured in osteogenic differentiation medium or osteogenic differentiation medium treated with 1 ?g/ml SPA for 2 weeks.The significantly differential lncRNA,closely related to osteogenesis differentiation,were verification by qRT-PCR to screen out the target gene.3.The effect of different lncRNA on the SPA-induced hBMSC sosteogenic differentiationThe three siRNA(siRNA-1,siRNA-2,and siRNA-3)targeting NONHSAT009968 was obtained.Approximately 200 nM siRNA was transfected into hBMSCs using Lipofectamine 2000 reagent according to the manufacturer's instructions.The efficiency of siRNA was determined by qRT-PCR.We selected siRNA-2 for lentivirus packaging.NONHSAT009968 shRNA lentiviral particles were generated.Lentivirus production,concentration,titration,infection conditions,and stable cell line selection were performed according to standard procedures.After infection with the lentivirus expressing NONHSAT009968-shRNA or empty lentivirus for 3 days,hBMSCs were harvested for qRT-PCR to confirm that the shRNA could successfully silence NONHSAT009968.After infection with lentivirus expressing NONHSAT009968-shRNA or empty lentivirus for 3 days,hBMSCs were cultured for 2 and 3 weeks.Calcium nodules deposition were detected by alizarin red staining.The level of IL-1a,IL-6,TNF-a,and ALP were detected by ELSIA.The expression of Runx2,OCN,OPN,and COL1 Alwere assayed by western blot.4.The mechanism about that the effect of different lncRNA on the SPA-induced hBMSC sosteogenic differentiationAfter infection with lentivirus expressing NONHSAT009968-shRNA or empty lentivirus for 3 days,hBMSCs were cultured for 2 and 3 weeks.The expression of Wnt3a,?-catenin,and GSK-3? were assayed by qRT-PCR and western blot to analyzed the effect of NONHSAT009968 interference on the the expression of Wnt3a,?-catenin,and GSK-3?.Results:1.The SPA-induced osteomyelitis model in vitro were established successfullySPA treatment could induce secretion of inflammatory cytokines IL-1a,IL-6,and TNF-a(P<0.05).The secretion of inflammatory cytokines IL-1a,IL-6,and TNF-a wasdose-dependent(0,0.1,0.5,1,10,100 ?g/ml).In addition,the alizarin red staining revealed that the five different concentrations of SPA could suppress calcium mineralization(P<0.05),with the degree of inhibition directly correlated to the SPA concentration.Thus,the results of ALP detection showed that SPA treatment could suppress ALP activity in a dose-dependent manner(P<0.05).The results showed that 1 ?g/ml SPA could significantly increase the secretion of IL-1a,IL-6 and TNF-a,inhibit calcium nodule deposition and ALP expression,and SPA(1 ?g/ml)-induced osteomyelitis model in vitro were established successfully.2.The analysis of the IncRNA microarray results(1)hBMSCs were cultured in osteogenic differentiation medium with 1 ?g/mL SpA for 2 weeks and cells were harvested for IncRNA microarray assays.We identified 2033 IncRNAs with aberrant expression in SpA-treated hBMSCs compared to controls.Among these IncRNAs,641 were down-regulated and 1392 were up-regulated(fold change>2.0,P<0.05)compared to controls.From the mRNA expression profiling data,449 mRNAs were identified as differentially expressed between the two groups.Among these mRNAs,318 were obviously down-regulated and 131 were obviously up-regulated(fold change>2.0,P<0.05).(2)Based on KEGG pathway analysis,the up-regulated coding mRNAs encompass 20 different pathways,and the most enriched pathways were salivary secretion,bile secretion,glutamatergic synapse,olfactory transduction,and insulin secretion.The down-regulated transcripts corresponded to 20 pathways,and the most enriched were hematopoietic cell lineage,retinol metabolism,drug metabolism,cell adhesion molecules,and metabolism of xenobiotics by cytochrome.To understand the functions of differentially expressed genes,all the differentially expressed genes were mapped to terms in the GO database and compared with the background.Based on the GO analysis for biological processes,the up-regulated transcripts were highly enriched for plasminogen activation,and water transport,while the down-regulated transcripts were highly enriched for cellular responses to glucocorticoid stimulus,and cellular glucuronidation.In the GO analysis for molecular function,we found that the up-regulated transcripts were highly enriched for neurotransmitter transporter activity,and calmodulin binding,whereas down-regulated transcripts were highly enriched for ligand-dependent nuclear receptor transcription coactivitor activity,and glucuronosyltransferase activity.(3)Through cis-100k analysis,five significantly differential lncRNAs related to osteogenic differentiation were identified,including NONHSAT125464,ENST00000504555,NONHSAT098635,NONHSAT054627,and NONHSAT009968.Their potential cis-regulated mRNA targets respectively included bone morphogenetic protein 1(BMP1),SMAD family member 1(SMAD1),SMAD1,collagen type ? alpha 1(COL1 A1),and Wnt family member 3A(Wnt3a),which have been demonstrated to function as regulators of osteoblast differentiation,or osteogenic differentiation markers.The expression of the 5 lncRNAs potentially related to osteogenic differentiation based on cis-100k analysis was verified by qRT-PCR.The results of qRT-PCR demonstrated that the expression levels of NONHSAT 125464,ENST00000504555,NONHSAT098635,NONHSAT054627,and NONHSAT009968 were obviously increased in the SPA treatment group compared with the normal culture group,which is consistent with the micro array experiment data.The expression of NONHSAT009968 exhibited the greatest increase.3.NONHSAT009968-silencing reversed the effect of SPA-inhibited hBMSC sosteogenic differentiationAmong the siRNAs tested,transfection with siRNA-2 and siRNA-3 could inhibit the expression of NONHSAT009968(P<0.05),siRNA-2 resulted in the lowest expression of NONHSAT009968,and was thus the best for silencing this IncRNA target.After infecting cells with lentivirus expressing NONHSAT009968-shRNA or NC,the expression level of NONHSAT009968 was reassessed by qRT-PCR.The results showed that NONHSAT009968 was successfully silenced via infection with the lentivirus.NONHSAT009968 shRNA or NC hBMSCs cultured in osteogenic differentiation medium with or without 1 ?g/mL SpA for 14 days or 21 days.The alizarin red staining showed that NONHSAT009968 silencing increased calcium mineralization by the cells compared to the NC group at both 14 days and 21 days(P<0.05).The results of ALP detection showed that NONHSAT009968-silencing also increased ALP activity compared to NC group at both 14 days and 21 days(P<0.05).In addition,NONHSAT009968-silencing increased the protein expression levels of Runx2,OCN,OPN,and COL1A1(P<0.05),which are involved in osteogenic differentiation.All of these results indicate that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs.4.NONHSAT009968-silencing promoted the expression of Wnt3a,?-catenin and GSK-3?SPA-induced significantly inhibited the expression of Wnt3a,?-catenin and GSK-3p(P<0.05),which was time-dependent.In addition,the NONHSAT009968-silencing significantly increased the protein expression levels of Wnt3a,?-catenin and GSK-3?(P<0.05),which was time-dependent.The results were showed that the mechanism about NONHSAT009968-silencing promote osteogenic differentiation may be related to the expression of Wnt3a and ?-catenin and GSK-3?.Conclusion:(1)1 ?g/ml SPA could significantly increase the secretion of IL-1a,IL-6 and TNF-a,inhibit calcium nodule deposition and ALP expression,inhibit the expression of Wnt3a,?-catenin,and GSK-3?,and SPA(1 ?g/ml)-induced osteomyelitis model in vitro were established successfully.(2)Five significantly differential IncRNAs related to osteogenic differentiation weae identified,including NONHSAT125464,ENST00000504555,NONHSAT098635,NONHSAT054627,and NONHSAT009968.The expression of NONHSAT009968 exhibited the greatest increase.NONHSAT009968 potential cis-regulated mRNA targets is Wnt3a,which have been demonstrated to function as regulators of osteoblast differentiation.(3)NONHSAT009968-silencing could significantly promote the expression of Wnt3a,?-catenin,and GSK-3?,increase the secretion of IL-la,IL-6 and TNF-a,promote calcium nodule deposition and ALP expression,increase the expression of Runx2,OCN,OPN,and COL1A1.NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs(4)In this study,the reason and mechanism of osteogenesis-reduced in osteomyelitis were firstly elucidated from the perspective of IncRNA,which provided a new explanation and research direction for future research... |
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