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Microarray profiling of circular RNAs in steroid-associated osteonecrosis of the femoral head: Observational study.

ABSTRACT: The aim of this study was to elucidate the molecular mechanisms and to identify the differential expression of circular RNAs (circRNAs) for steroid-associated osteonecrosis of the femoral head (SONFH) using bioinformatics analysis.circRNA microarray was performed with 3 SONFH tissues and the adjacent normal tissues, and differentially expressed circRNA were identified by limma package in R. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery database. In addition, a differentially expressed genes (DEG)-associated circRNA/microRNA (miRNA) interaction was predicted by combination of TargetScan and miRanda, and the circRNA/miRNA interaction network generated by the cytoscape software.A total of 647 differentially expressed circRNAs, including 433 upregulated and 214 downregulated circRNA were identified. The most enriched GO terms for upregulated and downregulated circRNA were extracellular matrix organization and leukocyte activation in biological process; extracellular matrix and spindle pole in cellular component; integrin binding and ATP binding in molecular function, and KEGG pathway enrichment analyses showed that the upregulated and downregulated circRNA were strongly associated with Protein digestion and absorption and Cell cycle. Moreover, a total of 212 differentially expressed messenger RNAs (mRNAs), including 113 upregulated and 99 downregulated genes were identified. In addition, from the analysis of miRNA, long noncoding RNAs, mRNA, and circRNA networks, we found that hsa_circ_0008136 and hsa_circ_0074758 were respectively the upregulated and downregulated circRNA with highest degrees.The identified circRNA and mRNA could be implicated in the progression of human SONFH. The findings could lead to a better understanding of SONFH pathogenesis.

SUBMITTER: Yao T

PROVIDER: S-EPMC7478599 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Microarray profiling of circular RNAs in steroid-associated osteonecrosis of the femoral head: Observational study.

Yao Tao T Yin Zong-Sheng ZS Huang Wei W Ding Zhen-Fei ZF Cheng Chao C

Medicine 20200301 10

The aim of this study was to elucidate the molecular mechanisms and to identify the differential expression of circular RNAs (circRNAs) for steroid-associated osteonecrosis of the femoral head (SONFH) using bioinformatics analysis.circRNA microarray was performed with 3 SONFH tissues and the adjacent normal tissues, and differentially expressed circRNA were identified by limma package in R. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were per ...[more]

PMID: 32150104

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Project description:BackgroundThe etiology and pathology osteonecrosis of the femoral head (ONFH) are not completely clarified. As a cytokine participating in systemic inflammation, tumor necrosis factor alpha (TNFα) has been shown to be involved in the pathogenesis of ONFH. However, the role of TNFα in ONFH is not clearly clarified. In the present study, we investigated the effects of TNFα on proliferation, angiogenesis, and osteogenic differentiation of rat bone mesenchymal stem cells (rMSCs) and the underlying mechanisms.MethodsAll femoral bone tissues were separated in surgeries. After extracting total RNA and protein, we evaluated TNFα content by ELISA and the relative expression levels of genes by quantitative real-time PCR and western blot. Also, immunohistochemistry staining was performed to observe the expression of Runx2 in the bone samples. Chick embryo chorioallantoic membrane (CAM) assay was performed to observe the effect of TNFα on angiogenesis. The genomic DNAs were treated by bisulfite modification, and methylation status of CpG sites in the CpG islands of human and rat Runx2 gene promoter was determined by DNA sequencing. The binding of H3K4me3 and H3K27me3 in Runx2 promoter was checked by ChIP assay. RNA-seq analysis was used to find out the genes and pathways changed by TNFα in rMSCs.ResultsThe results demonstrate TNFα promotes cell proliferation and angiogenesis whereas inhibits osteogenesis. Epigenetic regulations including DNA methylation and histone modifications play important roles in mediating the effect of TNFα on osteogenic differentiation. We find an increased rate of CpG methylation in rat Runx2 promoter in TNFα-treated rMSCs, as well as significantly increased occupancy of H3K27me3 in Runx2 gene promoter. The content of TNFα in necrotic tissue is much lower than that of normal tissue. And relevantly, human Runx2 promoter is demethylated in necrotic tissue using bone samples from patient with ONFH. In addition, we have observed that Wnt signaling pathway is inhibited by TNFα as multiple Wnts are markedly decreased in TNFα-treated rMSCs by RNA-seq analysis.ConclusionTaken together, our study shows that TNFα plays complicated roles in the pathogenesis of ONFH, including proliferation, angiogenesis, and osteogenesis. Targeting TNFα should not be considered as an applicable strategy to inhibit the progression of ONFH.

Project description:PurposeSteroid-induced necrosis of the femoral head (SONFH) is a refractory orthopedic hip disease occurring in young and middle-aged people, with glucocorticoids being the most common cause. Previous experimental studies have shown that cell pyroptosis may be involved in the pathological process of SONFH, but its pathogenesis in SONFH is still unclear. This study aims to screen and validate potential pyroptosis-related genes in SONFH diagnosis by bioinformatics analysis to further elucidate the mechanism of pyroptosis in SONFH.MethodsThere were 33 pyroptosis-related genes obtained from the prior reviews. The mRNA expression was downloaded from GSE123568 dataset in the Gene Expression Omnibus (GEO) database, including 10 non-SONFH (following steroid administration) samples and 30 SONFH samples. The pyroptosis-related differentially expressed genes involved in SONFH were identified with "affy" and "limma" R package by intersecting the GSE123568 dataset with pyroptosis genes. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the pyroptosis-related differentially expressed genes involved in SONFH were conducted by "clusterProfiler" R package and visualized by "GOplot" R package. Then, the correlations between the expression levels of the pyroptosis-related differentially expressed genes involved in SONFH were confirmed with "corrplot" R package. Moreover, the protein-protein interaction (PPI) network was analysed by using GeneMANIA database. Next, The ROC curve of pyroptosis-related differentially expressed genes were analyzed by "pROC" R package.ResultsA total of 10 pyroptosis-related differentially expressed genes were identified between the peripheral blood samples of SONFH patients and non-SONFH patients based on the defined criteria, including 20 upregulated genes and 10 downregulated genes. The GO and KEGG pathway enrichment analyses revealed that these 10 pyroptosis-related differentially expressed genes involved in SONFH were particularly enriched in cysteine-type endopeptidase activity involved in apoptotic process, positive regulation of interleukin-1 beta secretion and NOD-like receptor signaling pathway. Correlation analysis revealed significant correlations among the 10 differentially expressed pyroptosis-related genes involved in SONFH. The PPI results demonstrated that the 10 pyroptosis-related differentially expressed genes interacted with each other. Compared to non-SONFH samples, these pyroptosis-related differentially expressed genes had good predictive diagnostic efficacy (AUC = 1.000, CI = 1.000-1.000) in the SONFH samples, and NLRP1 had the highest diagnostic value (AUC: 0.953) in the SONFH samples.ConclusionsThere were 10 potential pyroptosis-related differentially expressed genes involved in SONFH were identified via bioinformatics analysis, which might serve as potential diagnostic biomarkers because they regulated pyroptosis. These results expand the understanding of SONFH associated with pyroptosis and provide new insights to further explore the mechanism of action and diagnosis of pyroptosis associated in SONFH.

Project description:BackgroundCellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations.MethodsIn the present study, the bone marrow samples of patients with ONFH (n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation.ResultsThe morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head.ConclusionReprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.

Dataset Information

Microarray profiling of circular RNAs in steroid-associated osteonecrosis of the femoral head: Observational study.

Publications

Microarray profiling of circular RNAs in steroid-associated osteonecrosis of the femoral head: Observational study.

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