A Study On The Differential Expression Of CircRNAs In Steroid-induced Osteonecrosis Of The Femoral Head

Background Osteonecrosis of the femoral head is a common orthopedic disease caused by the destruction of the blood supply of the femoral head and the necrosis of the components of the femoral head,which leads to joint dysfunction and pain,It occurs frequently among young adults.As a result,a large number of patients eventually have to undergo joint replacement,which brings a heavy burden to the society.In China,the application of hormones is the primary cause of osteonecrosis of the femoral head.Because the pathogenesis of steroid-induced osteonecrosis of the femoral head(SONFH)is not clear,it affects the early diagnosis and treatment of patients.Although many scholars have put forward many theories after long-term research,including lipid metabolism disorder theory,osteogenic weakening theory of bone marrow mesenchymal stem cells,apoptosis theory and gene polymorphism and so on,the exact pathogenesis of SONFH is not completely clear,and further research is still needed,which is still a hot and difficult point.Some scholars have studied the pathogenesis of SONFH from the perspective of non-coding RNA,especially the relationship between micro RNA(miRNA)and SONFH,and have achieved some results.However,there is still a lack of research on the relationship between Circular RNA(CircRNA)and the mechanism of SONFH.Therefore,this study takes circ RNA as the cut-in point for the first time to screen tissue samples from the necrotic area of the femoral head in patients with SONFH through high-throughput microarray to explore the mechanism of circular RNA in the occurrence and development of SONFH.ObjectiveWith the help of high-throughput competitive endogenous RNA(CeRNA)chiptechnology,the expression profile of circular RNAs in SONFH patients was obtained,and the results were verified.Bioinformatics techniques were used to analyze and predict the target genes and signal pathways regulated by non-coding RNA.The current research efforts to study the related functions of differentially expressed circular RNA,explore its possible mechanism,and preliminarily reveal the CeRNA mechanism of circular RNA in the formation of SONFH,establish the basis for further elucidating the pathogenesis of SONFH,and finally provide scientific basis for finding potential molecular markers or therapeutic targets for early diagnosis of SONFH.MethodsIn this study,high-throughput CeRNA chip was used to detect three pairs of SONFH tissue(ONs)and adjacent normal tissue(NLs),and the differential gene expression profile was obtained.By means of bioinformatics,the expression profile characteristics of differential genes were studied,and cluster analysis and enrichment analysis were carried out.The tissue samples of 10 pairs of SONFH patients and adjacent control tissues were collected,and the 10 candidate differentially expressed genes were verified by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR).The circular structure of differentially expressed circRNA has been experimentally confirmed.The miRNAs binding to hsa＿circ＿0058122 and its downstream target gene molecules were predicted by bioinformatics.The model of human umbilical vein endothelial cell(HUVECs)treated with dexamethasone was established in vitro.In order to study the function of hsa＿circ＿0058122,over-expression vectors and three small interferenceRNA(si RNA)targeted to the backspliced site of has＿circ＿0058122were designed for hsa＿circ＿0058122,respectively,and the interference efficiency was detected by fluorescence quantitative PCR.Flow cytometry was used to detect the effect of hsa＿circ＿0058122 overexpression and interference on apoptosis of HUVECs cells in each group.The level of apoptotic protein of HUVECs cells in each group wasdetected by immunoblotting test(WB).The expression of hsa-miR-7974,human insulin-like growth factor binding protein-5(IGFBP5)in HUVECs cells was analyzed by fluorescence quantitative PCR,and the level of IGFBP5 protein was detected by WB.Dual-luciferase assay was performed to confirm the combination between hsa＿circ＿0058122 and hsa-miR-7974.The co-localization of hsa＿circ＿0058122 and hsa-miR-7974 was detected by fluorescence in situ hybridization(FISH).The expression of hsa-miR-7974 in surgical samples was detected by fluorescence quantitative PCR,and the correlation between the expression pattern of hsa＿circ＿0058122 and hsa-miR-7974 was analyzed.Results1.In this study,tissue samples from patients with SONFH were screened by CeRNA microarray.A total of 647 differentially expressed circ RNAs were identified(Fold change ≥ 2,P ≤ 0.05),including 433 up-regulated and 214 down-regulated circ RNAs.A total of 436 differentially expressed lnc RNAs were identified(Fold change ≥ 2,P ≤0.05),including 210 up-regulated lnc RNAs and 226 down-regulated lnc RNAs.In addition,222 differentially expressed m RNA were identified(Fold change ≥ 2,P ≤0.05),including 113up-regulated genes and 109 down-regulated genes.Through the network analysis of the co-expression of miRNA,m RNA and circ RNA,We found circ RNA is very closely related to miRNA and m RNA2.Fluorescence quantitative PCR verified the primary screening results of the microarray,and it was found that there were significant differences in the expression of6 circ RNAs molecules and 3 lnc RNAs molecules in 10 pairs of tissue samples(P ≤0.05).The results are consistent with the chip test results.The ring structure of six circ RNAs molecules were confirmed by cyclization verification experiments.3.Flow cytometry showed that overexpression of hsa＿circ＿0058122 in HUVEC cellscould increase the proportion of apoptosis,while interfering with hsa＿circ＿0058122could decrease the proportion of apoptosis.Under the condition of hormone treatment,the proportion of apoptosis was higher after overexpression of hsa＿circ＿0058122,but decreased after interfering with hsa＿circ＿0058122.In hormone-treated and over-expressed has＿circ＿005812 HUVEC cells,the expression of pro-apoptotic proteins Bax and cleaved caspase-3 was up-regulated,while the low expression of apoptotic protein Bcl-2 was down-regulated.In the group of HUVEC cells interfering with has＿circ＿0058122,the expression of pro-apoptotic proteins Bax and cleaved caspase-3 was low,and the expression of anti-apoptotic protein Bcl-2 was increased(P≤ 0.05).The results showed that both hormone treatment and overexpression of has＿circ＿0058122 could induce apoptosis in HUVEC cells,while knockdown of has＿circ＿0058122 could inhibit the apoptosis of HUVEC cells.4.Based on the results of ceRNA network analysis,we predict that hsa＿circ＿0058122can competitively combine with miR-7974.The expression of miR-7974 was low in the necrotic area of the femoral head in 10 patients.The expression was highly expressed in 10 matched normal bone tissues,and the difference was statistically significant(P ≤ 0.05).The expression pattern of miR-7974 is opposite to that of hsa＿circ＿0058122 in tissue samples.The results of Dual-luciferase assay showed that there was interaction between hsa-miR-7974 and hsa＿circ＿00058122.In the FISH experiment,it was observed that the hybridization signals of hsa＿circ＿0058122 and miRNA were co-located,and both hsa＿circ＿0058122 and miR-7974 were located in the cytoplasm.5.The expression of hsa-miR-7974 was significantly down-regulated in hormone-treated and hsa＿circ＿00058122-overexpressed HUVEC cells,up-regulated in hsa＿circ＿00058122-knockdown HUVEC cells,and down-regulated in hormone-treated HUVEC cell model.Knockdown hsa＿circ＿00058122,hsa-miR-7974 expression was significantly up-regulated in hormone-treated HUVEC cell model.In summary,theexpression pattern of hsa＿circ＿00058122 in cells was opposite to that of hsa-miR-7974.Hormone treatment or high expression of hsa＿circ＿00058122 could down-regulate the expression of hsa-miR-7974.6.The predicted has-miR-7974 downstream target gene IGFBP5,was detected by qPCR and Western.The results showed that the expression of IGFBP5 was up-regulated in hormone-treated group and hsa＿circ＿0058122 overexpression group(p≤0.05),while the expression of IGFBP5 was down-regulated in HUVEC cell group interfering with hsa＿circ＿0058122(p≤0.05).In the hormone-interfered cell model,the expression of IGFBP5 had no significant change compared to the HUVEC blank cell group after interfering with hsa＿circ＿0058122(p>0.05).ConclusionThis study reported the differential gene expression profile of circ RNA in the tissues of patients with SONFH for the first time.The differential expression of 6 circular RNA molecules was verified in tissue samples,which showed that there was a correlation between circ RNA and the occurrence and development of SONFH.It has been found that hsa＿circ＿0058122 can adsorb hsa-miR-7974 and induce early apoptosis.It is preliminarily confirmed that hsa＿circ＿0058122 regulates the expression of IGFBP5 through ceRNA mechanism.It is revealed that the related genes may cause osteonecrosis of the femoral head by inducing the apoptosis of vascular endothelial cells.We speculate that IGFBP5 is a potential target for SONFH therapy.The mechanism of the differentially expressed circular RNA and predicted related ceRNA molecules in the pathogenesis of SONFH found in this research is worthy of more in-depth study.