Screening Of Differential Genes And Bioinformatics Analysis Of Osteonecrosis Of Femoral Head With Kidney Deficiency And Blood Stasis Syndrome Based On RNA-seq Technique

Objective1.Bioinformatics method was used to analyze the GEO microarray data to explore the differential gene expression profile and related molecular mechanism of steroid-induced osteonecrosis of the femoral head.2.Through the method of transcriptome high-throughput sequencing,to screen the biomarkers related to the diagnosis and treatment of osteonecrosis of the femoral head with kidney deficiency and blood stasis syndrome and to explore the molecular regulation mechanism of differentially expressed genes in the development of osteonecrosis of the femoral head.3.To study the effect of differentially expressed genes on osteogenic differentiation of bone marrow mesenchymal stem cells.Method1.Differential Gene expression profile and related Molecular Mechanism of steroid-induced Necrosis of the femoral head based on GEO Database.GEO2R was used to analyze the gene differential expression of the steroid-induced osteonecrosis of the femoral head selected from the comprehensive gene expression database(GEO).STRING-db database is used to construct protein-protein interaction network(PPI)and screen key proteins by R language software;Gene Ontology functional enrichment analysis is carried out by DAVID database;finally,DAVID database is used to realize signaling pathway enrichment analysis based on Kyoto encyclopedia of genes and genomes.2.Study on differentially expressed genes and their regulatory mechanism of necrosis of femoral head with kidney deficiency and blood stasis syndrome based on RNA-seq technique(1)With peripheral blood mononuclear cells as the research objects,4 patients with femoral head necrosis of kidney deficiency and blood stasis type(SIONFH group),4 patients with SLE without femoral head necrosis(SLE group),and 4 normal subjects(control group)were selected respectively.High-throughput transcriptome sequencing technology(RNA-seq)was used to screen the differentially expressed genes in each group.(2)STRING database was used to analyze the interaction network of differentially expressed genes and proteins,and bioinformatics methods were used to predict and analyze differentially expressed genes and their possible participation in the biological process and related signal pathways of kidney deficiency and blood stasis ONFH.3.Effect of differentially expressed genes on osteogenic differentiation of bone marrow mesenchymal stem cells in osteonecrosis of femoral head.The main results are as follows:(1)Bone marrow mesenchymal stem cells of SD rats were isolated,extracted and cultured,and the cells of the third generation were selected for experiment.(2)Kdm5d gene silencing plasmid was constructed,and rBMSCs cells were transfected with adeno-associated virus to observe the effect of silencing Kdm5d expression on osteogenic differentiation of rBMSCs in SD rats.The observation contents included alkaline phosphatase activity detection,alizarin red staining,fluorescence quantitative analysis of osteogenesis-related gene expression and so on.Result1.Differential Gene expression profile and related Molecular Mechanism of steroid-induced Necrosis of the femoral head based on GEO Database.(1)GEO microarray data analysis showed that compared with the control group,there were 1836 differentially expressed genes in peripheral blood serum of SIONFH patients,of which 1088 genes were up-regulated and 748 down-regulated.Core protein genes include ITGAM,TLR4,MAPK3,PTPRC,STAT3,TLR2,CXCL8,TLR8 and so on.Differentially expressed genes are involved in osteoclast differentiation,nuclear transcription factor NF-kappa B signal pathway,Toll-like receptor signal pathway,NOD-like receptor signal pathway,HIF-1 signal pathway and so on.2.Study on differentially expressed genes and their regulatory mechanism of necrosis of femoral head with kidney deficiency and blood stasis syndrome based on RNA-seq techniqueThe main results were as follows:(1)Bioinformatics analysis showed that there were 169 differentially expressed genes between SIONFH group and control group,including 38 up-regulated genes and 131 down-regulated genes.There were 74 differentially expressed genes between SIONFH group and SLE group,with 31 up-regulated genes and 43 down-regulated genes.(2)The results of GO enrichment analysis showed that the differentially expressed genes in the SIONFH group and the control group were involved in biological processes such as immune response,innate immune response,inflammatory response,etc.Differentially expressed genes in SIONFH group and SLE group regulate biological processes such as oxygen transport,immune response,inflammatory response,antigen processing and presentation,etc.(3)KEGG enrichment analysis showed that the differential genes in SIONFH group were involved in a variety of signal pathways,including TNF signal pathway,NF-kappa B signal pathway,type I diabetes signal pathway,phagosome signaling pathway and so on.The differential genes in SIONFH group and SLE group are involved in the regulation of chemokine signaling pathway,type I diabetes signaling pathway,rheumatoid arthritis signaling pathway and so on.3.Effect of differentially expressed genes on osteogenic differentiation of bone marrow mesenchymal stem cells in osteonecrosis of femoral head.(1)Compared with the blank control group,the alizarin red staining area in the Kdm5d#1 group decreased significantly(P<0.05).Compared with the blank control group,the alizarin red staining area in the Kdm5d#2 group decreased significantly(P<0.05).The alizarin red staining area in the Kdm5d#1 group was significantly lower than that in the Kdm5d#2 group(P<0.05).(2)The results of ALP staining showed that the number of positive staining cells in Control group was the most and the staining was the deepest,followed by Kdm5d#2 group,and the number of positive staining cells in Kdm5d#1 group was less than that in the blank control group.(3)Compared with control group,the expression of Kdm5d,Runx2,Opn,Ocn and Alp of bone marrow mesenchymal stem cells in Kdm5d#1 group decreased significantly(P<0.05).Compared with the control group,the expression of Kdm5d,Opn and Ocn in bone marrow mesenchymal stem cells in Kdm5d#2 group decreased significantly(P<0.05),while the expression of Alp and Runx2 in bone marrow mesenchymal stem cells in Kdm5d#2 group had no significant difference compared with the blank control group(P>0.05).Conclusion1.Multiple key differentially expressed genes regulate multiple signaling pathways to mediate the occurrence of SIONFH,and some key genes may be new biomarkers for the diagnosis of SIONFH or potential therapeutic targets,further revealing the complex molecular mechanism of action in the pathogenesis of SIONFH.2.Many key genes and various signaling pathways may play an important role in the process of femoral head necrosis caused by kidney deficiency and blood stasis.The results may provide an important theoretical basis for the early prevention,diagnosis and treatment of ONFH with kidney deficiency and blood stasis syndrome,or the research and development of targeted drugs.3.The expression of Kdm5d gene significantly reduced the mineralized nodules and alkaline phosphatase activity of rat bone marrow mesenchymal stem cells,and down-regulated the expression of Runx2,Opn,Ocn and Alp of BMSCs.Kdm5d may affect the osteogenic differentiation of BMSCs by regulating the expressions of Runx2,Opn,Ocn and Alp.