Role And Mechanism Of FAR591-mediated Apoptosis Of Bone Microvascular Endothelial Cells In The Pathogenesis Of Steroid-induced Osteonecrosis Of The Femoral Head

Objective:The specific pathogenesis of steroid-induced osteonecrosis of the femoral head（SONFH）is still not fully understood,and there is currently no exact and effective early cure.An in-depth study of the pathogenesis of SONFH has important practical significance for the early prevention and treatment of SONFH.Long non-coding RNA（lnc RNA）is involved in the pathogenesis and progression of various diseases by regulating gene expression;however,the role of lnc RNA in the pathogenesis and progression of SONFH requires further study.Therefore,this project sought to study the role and mechanism of Fos-associated linc RNA ENSRNOT00000088059.1（FAR591）-mediated apoptosis of bone microvascular endothelial cells（BMECs）in the pathogenesis of SONFH,which add new evidence for revealing the pathogenesis of SONFH and explore new targets for its early prevention and treatment.Methods:1.BMECs and osteoblast（OB）were isolated from Sprague-Dawley（SD）rats and cultured.Apoptosis of BMECs and OB were induced by different concentrations of hydrocortisone（HC）to compare the sensitivity of BMECs and OB to glucocorticoid（GC）.The SONFH model of SD rats was established by methylprednisolone（MP）.At different time points（2 and 4 weeks）after MP treatment,the expression levels of CD31（a marker of BMECs）and osterix（OSX,a marker of OB）were detected by immunofluorescence,BMECs apoptosis was detected by CD31 immunofluorescence combined with Td T-mediated d UTP nick-end labeling（TUNEL）,the microvascular volume and blood flow of the femoral head were detected by microfil angiography,and femoral head necrosis was detected by magnetic resonance imaging（MRI）,micro-computed tomography（micro-CT）,and hematoxylin-eosin（HE）staining,which investigated the role of GC-induced apoptosis of BMECs in the pathogenesis and progression of SONFH.2.GC concentration-dependent lnc RNAs and m RNAs were screened by the lnc RNA/m RNA microarray in BMECs apoptosis model induced by different concentrations of GC.These differentially expressed m RNAs were analyzed by the Kyoto Encyclopedia of Genes and Genomes（KEGG）and Gene Set Enrichment Analysis（GSEA）to select the core genes of the mitochondrial apoptotic pathway.The candidate lnc RNA-FAR591 was found to be related to apoptosis through the adjacent gene screening and coding/non-coding gene co-expression（CNC）analysis.The full-length sequence of FAR591 was detected by the rapid amplification of c DNA end（RACE）,the protein-coding ability of FAR591 was verified by pc DNA4/myc-His expression plasmid,the subcellular localization of FAR591 was detected by RNA-fluorescence in situ hybridization（RNA-FISH）.The microarray data was verified by real-time quantitative polymerase chain reaction（q PCR）,and the enrichment of glucocorticoid receptor（GR）on the promoter of the FAR591 gene was detected by chromatin immunoprecipitation（Ch IP）.It was screened and identified the lnc RNA-FAR591 related to GC-induced apoptosis of BMECs.3.Overexpression of FAR591 was performed by gene transfection,and knockout of the FAR591 gene was performed by the CRISPR/Cas9 technique.Subsequently,apoptosis of BMECs was induced by GC,TUNEL/4’,6-diamidino-2-phenylindole（DAPI）and Annexin V/propidium iodide（PI）were used to detect the apoptosis of BMECs,Western Blot was used to detect the expression of apoptosis-related proteins such as Bcl-2 interacting mediator of cell death（Bim）,P53 up-regulated modulator of apoptosis（Puma）,and Cleaved-Caspase3（Cleaved-CASP3）,which studied the role of FAR591 in GC-induced apoptosis of BMECs.4.Microarray was used to detect the gene expression profile in BMECs with FAR591 overexpression or knockout to screen the downstream targets that may be regulated by FAR591.The microarray data were verified by q PCR and Western Blot.The microarray screening data and verification results confirmed that Fos is the downstream target of FAR591 regulation.The expression of Fos in BMECs was intervened by gene overexpression or the CRISPR/Cas9 technique,and then,BMECs apoptosis was induced by GC.the expression of apoptosis-related proteins,including Bim,Puma,and Cleaved-CASP3,was detected by Western Blot,and the BMECs apoptosis was detected by TUNEL/DAPI and Annexin V/PI.It was studied the role of Fos in GC-induced apoptosis of BMECs.5.Blocking and rescue experiments were designed.The gene transfection technique was used to overexpress FAR591,and then Fos was further knocked out based on FAR591 overexpression.Additionally,the CRISPR/Cas9 technique was used to knock out FAR591,and then Fos was further upregulated based on the knockout of FAR591.Finally,GC was used to induce BMECs apoptosis.The expression of apoptosis-related proteins,including Bim,Puma,and Cleaved-CASP3,was detected by Western Blot,and BMECs apoptosis was detected by Annexin V/PI.It was studied whether FAR591 mediated GC-induced apoptosis of BMECs by regulating the expression of Fos.6.It was confirmed that the regulatory effect of FAR591 on Fos occurred at the transcriptional level.Then,specific oligonucleotide probes were designed according to the FAR591 sequence,and the cis-acting elements or trans-acting factors interacting with FAR591 were screened by chromatin isolation using RNA purification（Ch IRP）combined with high-throughput sequencing（Seq）and mass spectrometry（MS）analysis.Ch IRP-PCR and Ch IRP-Western Blot were used to verify whether FAR591 could bind to the Fos gene promoter and TATA-box binding protein associated factor 15（TAF15）,and chromatin immunoprecipitation（Ch IP）was used to detect the binding sites of TAF15 and RNA polymerase II（Pol II）on the Fos gene promoter.Finally,based on overexpression or knockout of FAR591,the enrichment of TAF15 and Pol II to Fos gene promoter was detected by Ch IP,and the expression level of Fos was detected by q PCR and Western Blot.It was studied the mechanism of FAR591 regulating Fos gene expression.7.The femoral head BMECs of SD rats were transfected with the adeno-associated virus to overexpress or knock out FAR591 in vivo,and the SONFH model was established by MP.Four weeks after MP treatment,CD31 fluorescent antibody was used to label BMECs,RNAscope and immunofluorescence were used to detect the expression of FAR591,Fos,Bim,and Puma in BMECs,TUNEL staining was used to detect the apoptosis of BMECs,microfil angiography was used to evaluate microvessel volume and blood flow of the femoral head,immunofluorescence was used to detect the expression of runt-related transcription factor 2（Runx2）and OSX,and MRI,micro-CT,and HE staining were used to evaluate osteonecrosis,which investigated the role of FAR591-mediated BMECs apoptosis in the pathogenesis and progression of SONFH.Results:1.A low concentration of HC（0.20 mg/m L）induced high levels of BMECs apoptosis and inhibited tube formation.Induction of OB apoptosis and inhibition of osteogenesis required a higher concentration of HC（0.30 mg/m L）,which indicated that BMECs were more sensitive to GC than OB.The SONFH model was established by MP.Two weeks after MP treatment,the expression level of CD31 in the model group decreased,the rate of CD31⁺-TUNEL⁺double-positive cells increased,angiography showed that the subchondral microvascular volume and blood flow of the femoral head decreased.However,there was no significant change in the expression level of OSX,and no obvious signs of osteonecrosis by HE staining,MRI,and micro-CT.Four weeks after MP treatment,the expression level of CD31 in the model group continued to decrease,the rate of CD31⁺-TUNEL⁺double-positive cells further increased,and angiography showed that the subchondral microvascular volume and blood flow of the femoral head decreased significantly.At this time,the expression level of OSX decreased,and HE staining showed that the subchondral trabecular bone was disordered,thinner and interrupted,OB around the trabecular bone disappeared,and many empty bone lacunae were found.MRI（T1 and T2 weighted images）showed abnormal signals of osteonecrosis of the femoral head.micro-CT showed decreases in the bone mineral density（BMD）,bone volume fraction（BVF）,trabecular number（Tb.N）,and trabecular thickness（Tb.Th）（P<0.05）.These results showed that GC-induced apoptosis of BMECs was the pre-event of SONFH.2.A total of 104 GC concentration-dependent lnc RNAs and 345 GC concentration-dependent m RNAs were detected in BMECs by lnc RNA/m RNA microarray（Fold change>2,P<0.05）.KEGG and GSEA showed that these m RNAs were significantly enriched in the apoptosis signaling pathway,and were mainly genes of the mitochondrial apoptotic pathway.The core genes of the mitochondrial apoptotic pathway were analyzed by CNC analysis（PCC>0.99,P<0.01）and adjacent gene screening（distance<200 kb）,and the candidate lnc RNA-FAR591 related to apoptosis was identified.RACE analysis showed that the full-length of FAR591 was1041 bp.Bioinformatics analysis and pc DNA4/myc-His expression plasmid confirmed that FAR591 did not encode protein.RNA-FISH showed that FAR591 was mainly located in the nucleus.In the model of GC-induced BMECs apoptosis,the expression of FAR591 was consistently up-regulated with the increase in GC concentration and apoptosis rate.Significantly increased expression of FAR591 was also detected in BMECs of the SONFH tissue.Ch IP-seq analysis showed that GR bound to the FAR591 gene promoter,and GC increased the enrichment level of GR on the FAR591gene promoter.These results indicated that FAR591 was related to GC-induced apoptosis of BMECs.3.The FAR591 gene in BMECs was successfully knocked out by the CRISPR/Cas9 technique.Following the knockout of the FAR591 gene,the expression of Bim,Puma,and Cleaved-CASP3 were down-regulated,and the apoptosis rate of BMECs was significantly reduced,which indicated that the knockout of FAR591effectively blocked the GC-induced apoptosis of BMECs.In contrast,FAR591 was successfully overexpressed in BMECs by the gene transfection technique.After the overexpression of FAR591,the expression of Bim,Puma,and Cleaved-CASP3 were up-regulated,and the apoptosis rate of BMECs was significantly increased,which showed that the overexpression of FAR591 significantly promoted GC-induced apoptosis of BMECs.These results confirmed that FAR591 mediated GC-induced apoptosis of BMECs.4.The downstream targets regulated by FAR591 were screened by microarray.The results showed that Fos was up-regulated after FAR591 overexpression and down-regulated after FAR591 knockout.Western Blot verified the microarray data,and the results further confirmed the regulatory effect of FAR591 on Fos expression.The Fos gene in BMECs was knocked out by CRISPR/Cas9 technique,and the expression of Bim,Puma,and Cleaved-CASP3 were down-regulated,which effectively blocked the GC-induced apoptosis of BMECs.However,Fos was overexpressed in BMECs using the gene transfection technique,and the expression of Bim,Puma,and Cleaved-CASP3 were up-regulated,which significantly promoted the GC-induced apoptosis of BMECs.These results showed that Fos,as the downstream regulatory target of FAR591,promoted GC-induced apoptosis of BMECs.5.The results of the blocking and rescue experiments showed that overexpression of FAR591 promoted the expression of Bim,Puma,and Cleaved-CASP3,and increased the apoptosis rate of BMECs.Knockout of Fos based on FAR591overexpression down-regulated Bim,Puma,and Cleaved-CASP3 expression,and decreased the apoptosis rate of BMECs,which indicated that knockout of Fos blocked the pro-apoptotic effect of FAR591.The knockout of FAR591 down-regulated the expression of Bim,Puma,and Cleaved-CASP3,and reduced the apoptosis rate of BMECs.Overexpression of Fos based on FAR591 knockout up-regulated the expression of Bim,Puma,and Cleaved-CASP3,and increased the apoptosis rate of BMECs,which confirmed that overexpression of Fos reversed the result of FAR591knockout.These results confirmed that FAR591 mediated GC-induced apoptosis of BMECs by regulating the expression of Fos.6.FAR591 was located in the nucleus.After overexpression or knockout of FAR591,the expression of Fos at both m RNA and protein levels was significantly changed,indicating that the regulation of FAR591 on Fos may occur at the transcriptional level.The screening results of Ch IRP-Seq showed that the FAR591-bound motif was AGRGGGYRWDMGGGAS.We identified 80523 enrichment peaks in the genome(P<10^-5),among which,4.35%were located in gene promoters,including the Fos gene promoter.The Ch IRP-PCR results also confirmed that FAR591 was bound to the Fos gene promoter and that the binding site was in the upstream region（-245～-51）of the transcription start site（TSS）of the Fos gene.Ch IRP-MS identified 27 proteins that specifically bind to FAR591,which are related to transcriptional activation,including TAF15.MS detected four specific peptides in TAF15,with a sequence coverage of 21.20%.The Ch IRP-Western Blot verification results also confirmed that FAR591 combined with TAF15.The Ch IP results showed that TAF15 and Pol II bound to the promoter of the Fos gene and that the binding site was near TSS（-108～+80）.Overexpression of FAR591 increased the enrichment of TAF15 and Pol II to Fos gene promoter,which in turn promoted Fos gene expression.Knockout of FAR591 reduced the enrichment of TAF15 and Pol II to the Fos gene promoter,which in turn suppressed Fos gene expression.These results showed that FAR591 recruited TAF15 and Pol II in the promoter region of the Fos gene,and then promoted the expression of the Fos by transcriptional activation.7.FAR591 gene was knocked out in femoral head BMECs,the expression of Fos,Bim,and Puma were down-regulated,the apoptosis rate of BMECs was decreased,the volume and blood flow of the femoral head microvessels were increased,and the expression of Runx2 and OSX were up-regulated.HE staining showed a reduction in empty bone lacunae.Moreover,micro-CT examination showed that the arrangement of subchondral trabeculae was still regular,BMD,BVF,Tb.N,and Tb.Th were increased,only a few femoral heads showed signs of osteonecrosis on MRI,and the incidence of SONFH was significantly reduced to 12.50%（P<0.01）.On the contrary,FAR591 was overexpressed in femoral head BMECs,the expression of Fos,Bim,and Puma were significantly up-regulated,the apoptosis rate of BMECs was increased,the volume and blood flow of femoral head microvessels were decreased,and the expression of Runx2 and OSX were significantly down-regulated.HE staining showed that the OB around the trabecular bone disappeared,and a large number of empty bone lacunae were observed.Moreover,micro-CT showed that the subchondral trabecular bone was widely absorbed and interrupted,BMD,BVF,Tb.N,and Tb.Th were significantly decreased,a large number of femoral heads showed signs of osteonecrosis on MRI,and the incidence of SONFH was significantly increased to93.33%（P<0.01）.These results confirmed that FAR591 mediated GC-induced apoptosis of BMECs,which led to the dysfunction of femoral head microcirculation and promoted the pathogenesis and progression of SONFH.Conclusions:In this study,we confirmed that GC-induced apoptosis of BMECs was the pre-event of SONFH.Simultaneously,we identified a new lnc RNA in BMECs,namely,FAR591—which responded to GR-mediated nuclear effects—was overexpressed following GC treatment of BMECs,and then mediated GC-induced apoptosis of BMECs.Mechanistically,FAR591 bound to the Fos gene promoter to form a stable RNA:DNA triplet structure.Subsequently,FAR591 recruited TAF15 and Pol II to promote the formation of a preinitiation complex in the Fos gene promoter,which promoted the expression of the Fos gene through transcriptional activation,thus activating the Fos/mitochondrial apoptotic pathway leading to BMECs apoptosis.Moreover,FAR591 mediated the GC-induced apoptosis of BMECs to cause femoral head microcirculation dysfunction,which then promoted the pathogenesis and progression of SONFH.