LncRNA NONHSAT009968 Inhibits The Osteogenic Differentiation Of HBMMSCs Induced By SPA Via Wnt3a

Background:Osteomyelitis is an infectious disease of the bone and medullary cavity that causes necrosis of bone and surrounding soft tissue.It is one of the most challenging diseases in the field of orthopedics,which can be caused by diseases such as trauma,surgery,diabetes and blood-borne infections.Patients often need to be hospitalized for a long time,have repeated operations,consume huge costs and use a large amount of antibiotics,have a high amputation rate and disability rate,bring more serious negative impact on patients' life and work,and produce greater socio-economic and psychological pressure.Although some progress has been made in surgical treatment,the mechanism of its occurrence and development is still unclear,its diagnosis process is complex,and the therapeutic effect of various methods is poor.Staphylococcus aureus(SA)is the most common pathogen causing osteomyelitis.It is believed that the mechanism of staphylococcus aureus causing osteomyelitis includes pathogenic factors and host factors.Staphylococcus aureus reaches the bone surface via iatrogenic or blood-derived pathways and can easily adhere to soft tissue,bone or metal implants.Bacteria can do this by recognizing microbial surface components that adhere to matrix molecules,such as collagen-binding proteins and bone saliva-binding proteins,and by binding to extracellular matrix(ECM)proteins.In recent years,with the development and deepening of epigenetics,more and more studies have shown that non-coding RNAs affect BMSCs osteogenic differentiation through various signaling pathways and regulatory factors.Although the role of protein-coding genes in BMSCs osteogenic differentiation has been extensively studied,the function of non-coding RNA in BMSCs osteogenic differentiation is still unclear.Non-coding RNAs are considered to play a key role in hBMMSCs osteogenic differentiation,including chromatin modification,transcription factor binding,endogenous capacity mechanisms and other post-transcription mechanisms.Long non-coding RNA(LncRNA)is a new class of non-coding transcripts with a length of more than 200 nucleotides.Recently,a number of studies have shown that LncRNAs are widely involved in growth and development by controlling the fate of cells(including MSC),and an increasing number of LncRNAs promote or inhibit the osteogenic differentiation of hBMMSC through a variety of regulatory factors and signaling pathways.Previous studies in this study have shown that lncRNA NONHSAT009968 can inhibit the osteogenic differentiation of hBMMSCs under the action of SPA,but its regulatory mechanism remains unclear.In the early stage of this project,cis-100K analysis showed that Wnt3a was one of the target genes of lncRNA NONHSAT009968,and the addition of SPA treatment in the osteogenic induction process of hBMMSCs could inhibit the expression of Wnt3a,while the inhibition of the expression of lncRNA NONHSAT009968 could promote the expression of Wnt3a.Therefore,we speculated that the mechanism of osteogenic differentiation of hBMMSCs regulated by lncRNA NONHSAT009968 may be related to the expression of Wnt3a.Objectives:(1)In vitro,to investigate whether Wnt3a affects the osteogenic differentiation of hBMMSCs under SPA by constructing hBMMSCs of interfered and overexpressed Wnt3a.(2)In vitro,to explore whether lncRNA NONHSAT009968 affects the osteogenic differentiation of hBMMSCs under SPA through Wnt3a by constructing Wnt3a-interfered and lncNONHSAT009968-interfered hBMMSCs.(3)To investigate the effect of lncRNA NONHSAT009968 on osteogenic differentiation and bone defect repair ability of hBMMSCs under the condition of osteomyelitis by constructing an animal model of chronic osteomyelitis.(4)The luciferase assay was used to analyze how lncRNANONHSAT009968 regulates the transcription level of Wnt3a by binding with the cis-acting elements on the Wnt3a promoter,and to clarify the regulatory mechanism of IncRNA NONHSAT009968 on the expression of Wnt3a.Through the above in vivo and in vitro experiments,the role and possible regulatory mechanism of LncNONHSAT009968 on osteogenic differentiation and bone defect repair ability of hBMMSCs under infection environment were revealed,and the experimental basis and new ideas were provided for the diagnosis and treatment of osteomyelitis and infectious bone defect.Methods:1.Cell level study on the influence of Wnt3a on the osteogenic differentiation ability of hBMMSCs under SPA stimulus.Wnt3a vector was constructed to overexpress lentivirus,and lentivirus was packaged.The expression of Wnt3a was detected by QRT-PCR and Western blot after hBMMSCs were infected.The virus-infected hBMMSCs were continuously stimulated by SPA(1?g/mL)and osteogenic induction was conducted for 7,14,and 21 days.Osteo-genic related genes COL1A1,OCN,OPN,Runx2,Wnt3a,?-catenin and GSK-3? were detected.Alizarin red staining and ALP were detected to assess osteogenic induction.2.Cell level study of lncRNA NONHSAT009968 influenced the osteogenic differentiation of hBMMSCs under SPA stimulus through Wnt3a.Wnt3a sh-RNA lentivirus vector was constructed,and lentivirus was packaged.The expression of Wnt3a was detected by quantitative PCR and Western blot after infection with hBMMSCs/NONHSAT-009968 sh-RNA.hBMMSCs infected with Wnt3a sh-RNA and NONHSAT009968 sh-RNA virus were continuously treated by SPA(1?g/mL),and osteogenic induction was conducted for 7,14,21 days.Detection of osteogenic related genes COL1A1,OCN,OPN,Runx2 Wnt3a,?-catenin,GSK-3? were performed.Alizarin red staining and ALP were detected to assess osteogenic induction.3.Animal level analysis of the effect of lncRNA NONHSAT009968 on osteogenic differentiation of hBMMSCs in osteomyelitis state.Rabbit osteomyelitis with bone defect model was established,and the effect of silencing lncRNA NONHSAT 009968 hBMMSCs on bone formation was proved by in vivo experiments.Gross observation,histomorphology and micro-CT were used to evaluate the osteogenesis ability and the healing of the bone ends in each group qualitatively and quantitatively.The expression of osteogenic related genes COL1A1,OCN,OPN and Runx2 as well as Wnt3a,?-catenin,GSK-3? were detected by QRT-PCR and Western blot.4.The research of molecular mechanism of targeted regulation of Wnt3a by lncRNA NONHSAT009968.The promoter sequence of Wnt3a was amplified by PCR,and a luciferase expression vector with deletion of the promoter sequence was constructed.The effect of overexpression of NONHSAT009968 on the activity of Wnt3a promoter with different lengths was detected by luciferase assay.The possible action sites of NONHSAT009968 on Wnt3a promoter were analyzed.Results:1.Over-expression of Wnt3a can significantly increase the expression of osteoblasts related genes COL1A1,OCN,OPN,Runx2,Wnt3a,?-catenin,while decrease the expression of GSK-3?,promote the expression of osteoblasts related proteins COL1A1,OCN,OPN,Runx2,Wnt3a,?-catenin,while decrease the expression of GSK-3?,promote the deposition of calcium nodules,increase the expression of ALP,and improve the osteogenic differentiation of SPA-inhibited hBMMSCs.2.Knockdown of both LncRNANONHSAT009968 and Wnt3a can significantly reduce the expression of osteogenic related genes COL1A1,OCN,OPN,Runx2,Wnt3a,(3-catenin,while increasing the expression of GSK-3?,reducing the expression of osteogenic related proteins COL1A1,OCN,OPN,Runx2,Wnt3a,?-catenin,while increasing the expression of GSK-3?,reducing the deposition of calcium nodules,and reducing the expression of ALP.Reverse the osteogenic differentiation of hBMMSCs induced by single knockdown LncRNANONHSAT009968 under SPA stimulus.3.For infectious bone defect repair,knockdown LncRNANONHSAT009968 in hBMMSCs and combined with hydroxyapatite composite scaffold can significantly improve osteogenesis related genes COL1A1,OCN,OPN,Runx2,Wnt3a,?-catenin expression,and reduce the expression of GSK-3?,promote osteogenesis related proteins COL1A1,OCN,OPN,Runx2,Wnt3a,?-catenin expression,reduce the expression of GSK-3?,promote calcium nodule deposit,and improve ALP expression.Micro-CT osteoblast related indexes and histopathological morphometrics osteoblast related indexes showed a encouraging outcome in promoting osteogenesis,but did not produce significant clinical repair effect.4.There was no significant difference in relative luciferase activity between the Wnt-NC+lncRNA-NC group and the Wnt-NC+lncRNA overexpression group,Wnt2+lncRNA-NC group and Wnt2+lncRNA overexpression group,and Wnt3+lncRNA-NC group and Wnt3+lncRNA overexpression group(P>0.05).There was significant difference in relative luciferase activity between Wntl+lncRNA-NC group and Wnt1+lncRNA overexpression group(P<0.05).Conclusions:1.Wnt3 a plays an important positive regulatory role in osteogenic differentiation of hBMMSCs stimulated by SPA in vitro.2.LncRNANONHSAT009968 plays a negative regulatory role in the osteogenic differentiation of hBMMSCs by inhibiting Wnt3a.3.LncRNANONHSAT009968 can inhibit bone formation during the osteogenesis of hBMMSCs under in vivo and in vitro infectious and inflammation conditions,which has the significance of being a potential therapeutic target.4.LncRNANONHSAT009968 may inhibits the transcription process of Wnt3a by binding to the promoter region of Wnt3a,thereby inhibiting the expression of Wnt3a and affecting the occurrence and development of osteomyelitis.