Gene Expression Profiling Of The Bone Trabecula In Patients With Osteonecrosis Of The Femoral Head And The Relevant Mechanism

Objective: Early diagnosis of osteonecrosis of the femoral head(ONFH)is challenging.Previous studies showed that bone trabecula play an important role in severity and progression of ONFH.Thus,gene expression profiling of bone trabecula is sequenced to investigate the genes alteration in ONFH patients.Methods: RNA sequencing was performed to identify gene expression profiling in osteonecrotic bone trabecula(ONBT),adjacent“normal’’ bone trabecula(ANBT)in five patients with ONFH,and bone trabecula in five patients with fracture of femoral neck,respectively.Then,we predicted the potential functions of these altered genes expression using Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.Additionally,the key candidate genes were detected by quantitative real-time polymerase chain reaction(QPCR)and western bolt(WB).Chromatin immunoprecipitation assays(ChIP)was used to verify the interaction between hif-1α and LCN2.Results: Histological results showed that osteonecrotic bone trabecula(ONBT)was characterized by necrosis,fibrosis and lacuna,but adjacent‘‘normal’’ bone trabecula(ANBT)was pathologically normal.RNA-seq data indicated that 12297 differentially abundant mRNAs(1032upregulated and 1265 downregulated)in ONBT group and 1523 differentially abundant mRNAs(744 upregulated and 799 downregulated)in ANBT group compared with healthy control group.Although ANBT showed no histological difference with the normal bone,the varied mRNA in ANBT was altered,partially similar with ONBT.GO enrichment analysis suggested that fatty acid metabolism and degradation were the main terms enriched with differentially expressed genes(DEG),and KEGG pathway enrichment analysis indicated PPARγ pathway was the most significantly regulated pathway.Interestingly,Lipocalin-2(LCN2),an osteoblast-enriched secreted protein decreased and it suggested that downregulation of LCN2 might affect lipid metabolism and lead to hyperlipemia,and thus promote pathogenesis of ONFH.Promoter sequence of the LCN2’s upstream was forecasted by bioinformatic method including the specific DNA-binding sequence of hif-1α.ChIP-QPCR showed that binding level of hif-1α with LCN2 promoter region in ANBT is higher than ONBT.Conclusion: These findings indicated,for the first time,LCN2 might play an important role in necrosis of bone trabecula and be served as diagnostic and therapeutic targets.