The Mechanism Of Icariin Regulating The Angiogenic Differentiation Of BMECs In Osteonecrosis Of Femoral Head Through LncRNA GAS5/miRNA-222 Pathway

BackgroundOsteonecrosis of femoral head(ONFH)is a devastating condition associated with apoptosis of the osteocytes and the bone marrow,which has a natural history of relentless progressive necrosis leading to fracture of subchondral bone plate,collapse of femoral head articular surface,and eventual premature osteoarthritis of hip.Among the etiologies,abuse of glucocorticoid is the most prominent causative factor of the ONFH.Studies showed that disfunction of bone microvascular endothelial cells(BMECs)played an important role in the pathophysiology of steroid-induced ONFH(SONFH).Epimedium is a traditional oriental herbal medicine,which possesses the function of tonifying liver and kidney,strengthening bones,and eliminating wind.Icariin(ICA)is a major flavonoid isolated from the epimedium.In vivo and in vitro experiments indicated that glucocorticoid inhibited BMECs apoptosis,migration,and tube formation,while ICA reversed this inhibition.However,the potential mechanism by which ICA alleviates SONFH still remains unclear.Our previous studies revealed that ICA could exert its biological activity in SONFH by regulating miRNAs expression.However,differential expression of miRNAs cannot adequately explain the pathophysiology of SONFH.Long non-coding RNA(IncRNA)can compete with miRNAs response elements for the same miRNA through the competitive endogenous RNA(ceRNA)mechanism,thereby regulating the expression of miRNA targeted genes.We hypothesized that ICA may regulated the differentiation of BMECs via lncRNA/miRNA singal pathway,and then alleviated SONFH.ObjectiveWe aimed to profile the different expression genes of BMECs from SONFH.Then according to the results of gene sequencing,we investigated whether ICA regulates BMECs'angiogenic differentiation through the IncRNA GAS5/miRNA-222 signal pathway.Methods1.Cancellous bone was harvested from the femoral heads of 5 SONFH patients and 5 control ones respectively during THA.BMECs were isolated by enzyme reaction and density gradient centrifugation methods.In addition,their phenotype was identified by immunofluorescence method.2.We used high-throughput sequencing to acquire the differentially expressed mRNAs,miRNAs,IncRNAs,and circRNAs in BMECs,after then we performed GO,pathway,and disease analyses about the differentially expressed genes,and co-expression analyses of IncRNA-miRNA-mRNA and ceRNA through bioinformatics analysis.3.Cell transfection and different treatments of BMECs using methylprednisolone(MPS)or mixture of MPS+ICA were performed.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miRNA-222;Western blot was used to perceive the protein levels of angiogenic differentiation markers,including basic fibroblast growth factor(bFGF),transforming growth factor-?(TGF-?),vascular endothelial growth factor(VEGF),and prostaglandin E(PGE);and Human VEGF ELISA kit was used to identify protein activity of VEGF.4.The interaction between IncRNA GAS5 and miRNA-222 was verified by RNA binding protein immunoprecipitation(RIP)and RNA pull-down experiments.The regulation of IncRNA GAS5 on miRNA-222 expression was observed by cell transfection experiments.After treatment of different transfected BMECs with MPS or mixture of MPS+ICA,the expressions of IncRNA GAS5,miRNA-222,and VEGF mRNA were detected by qRT-PCR;Western blot was used to detect the protein levels of bFGF,TGF-?,VEGF and PGE;and the Human VEGF ELISA kit was used to detect the activity of VEGF in BMECs.Results1.The cultured mature cells look like pebbles or paving stones under microscope.Immunofluorescence's identification show that vWF and CD31 are expressed in both groups,with a positive rate of 100%.These results display that the extracted cells possess the characteristics of vascular endothelial ones.2.Results of high-throughput sequencing revealed that there were 7856 mRNAs,386 miRNAs,1556 lncRNAs and 541 circRNAs differentially expressed in the SONFH group's BMECs.In addition,co-expression networks were established for differentially expressed IncRNAs-miRNAs-mRNAs and ceRNAs.The differentially expressed mRNAs mainly enrichment in biological processes including vasculature,angiogenesis,vascular morphology,cell migration,cell proliferation,and cell cycle.The differentially expressed miRNAs mainly enrichment in the regulation of metabolic processes,quality process,organic process of somatic cell,cell biology and signal pathways,cell differentiation,and cell biology.The differentially expressed circRNAs mainly enriched in the regulation of compounds containing nuclease metabolism,RNA metabolism,RNA polymerase ? transcription,and gene expression.The differentially expressed lncRNA-mRNA mainly enriched in regulation the orderly formation of nucleosomes and chromatin by histones,and the biological processes of DNA.These results indicated that mRNA is the executor of specific functions,while miRNA,lncRNA and circRNA are the functional regulators.3.Compared with MPS group,the inhibition of miRNA-222 group could significantly up-regulate the mRNA levels of bFGF,TGF-?,VEGF and PGE in BMECs,and increase the protein activity of VEGF in MPS+ICA group.Compared with MPS+ICA+mimic negative control(mimic-NC)group,the over-expression of miRNA-222 group could significantly down-regulate the mRNA levels of bFGF,TGF-?,VEGF and PGE in BMECs,and reduce the protein activity of VEGF in MPS+ICA+miRNA-222 mimic group.Compared with MPS+ICA+inhibitor-NC group,the inhibition of miRNA-222 group could significantly up-regulate the mRNA levels of bFGF,TGF-?,VEGF and PGE in BMECs,and increase the protein activity of VEGF in MPS+ICA+miRNA-222 inhibitor group.These results showed that ICA could promote BMECs' angiogenic differentiation through inhibition of miRNA-222.4.The results of RIP and RNA pull-down experiments showed that there was an interaction between lncRNA GAS5 and miRNA-222.Overexpression of lncRNA GAS5 inhibited the expression of miRNA-222,while its knockdown promoted miRNA-222 expression.Under the regulation of ICA,expression of miRNA-222 in BMECs was significantly up-regulated after interference with lncRNA GAS5,and mRNA level of VEGF and protein levels of bFGF,TGF-?,VEGF and PGE were significantly down-regulated and VEGF protein activity was significantly reduced,while these effects were reversed after the transfection of miRNA-222 inhibitor,suggesting that ICA regulated the expression of VEGF and the angiogenic differentiation of BMECs through the lncRNA GAS5/miRNA-222 singal pathway.ConclusionsICA down-regulated miRNA-222 by up-regulating lncRNA GAS5,and then up-regulated the downstream target molecule VEGF.ICA may promote the angiogenic differentiation of steroid-induced BMECs through the aforementioned signal pathway,ultimately defer the progress of SONFH.