Identification and validation of endoplasmic reticulum stress-related genes in patients with steroid-induced osteonecrosis of the femoral head.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a debilitating condition caused by long-term corticosteroid use, leading to impaired blood flow and bone cell death. The disruption of cellular processes and promotion of apoptosis by endoplasmic reticulum stress (ERS) is implicated in the pathogenesis of SONFH. We identified ERS-associated genes in SONFH and investigated their potential as therapeutic targets. We analysed the GSE123568 GEO dataset to identify differentially expressed genes (DEGs) related to ERS in SONFH. We conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, identified hub genes by protein-protein interaction (PPI) analyses, and evaluated their functions by gene set enrichment analysis (GSEA). We constructed mRNA-miRNA networks, identified potential therapeutics, and assessed immune cell infiltration. We performed cross-validation using the GEO dataset GSE74089, qRT-PCR on clinical samples from patients with SONFH and controls, and a receiver operating characteristic (ROC) curve analysis to assess the diagnostic performance of the hub genes. We identified 195 ERS-related genes in SONFH, which were primarily involved in oxidative stress, immune responses, and metabolic pathways. The PPI network suggested CXCL8, STAT3, IL1B, TLR4, PTGS2, TLR2, CASP1, CYBB, CAT, and HOMX1 to be key hub genes, which were shown by GSEA to be involved in biological pathways related to metabolism, immune modulation, and cellular integrity. We also identified 261 microRNAs (miRNAs) as well as drugs such as dibenziodolium and N-acetyl-L-cysteine that modulated inflammatory responses in SONFH. Twenty-two immune cell subtypes showed significant correlations, such as a positive correlation between activated mast cells and Tregs, and patients with SONFH had fewer dendritic cells than controls. The hub genes CYBB and TLR4 showed significant correlations with M1 macrophages and CD8 T cells, respectively. Cross-validation and qRT-PCR confirmed the upregulation of STAT3, IL1B, TLR2, and CASP1 in patients with SONFH, validating the bioinformatics findings. An ROC curve analysis confirmed the diagnostic potential of the hub genes. The top 10 hub genes show promise as ERS-related diagnostic biomarkers for SONFH. We discovered that 261 miRNAs, including hsa-miR-23, influence these genes and identified potential therapeutics such as dibenziodolium and simvastatin. Immune profiling indicated altered immune functions in SONFH, with significant correlations among immune cell types. Validation confirmed the upregulation of STAT3, IL1B, TLR2 and CASP1, which had diagnostic potential. The findings suggest potential diagnostic markers and therapeutic targets for SONFH.

Genetic Analysis of Potential Biomarkers and Therapeutic Targets in Ferroptosis from Steroid-Induced Osteonecrosis of the Femoral Head Based on Machine Learning.

OBJECTIVE: To locate the candidate therapeutic target genes involved in ferroptosis in steroid-induced osteonecrosis of the femoral head (SONFH). STUDY DESIGN: Bioinformatics analysis study. Place and Duration of the Study: Department of Orthopaedic Surgery, Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine, Guangdong, China, from March to July 2023. METHODOLOGY: After processing the gene expression omnibus (GEO) data with the R programming language, differentially expressed ferroptosis-related genes in SONFH were identified. To pinpoint the genes most strongly linked to SONFH in association with ferroptosis, least absolute shrinkage and selection operator (LASSO) regression and support vector machine-recursive feature elimination (SVM-RFE) were employed. Subsequently, the screened essential genes were analysed to investigate immune cell infiltration, and competing endogenous RNA (ceRNA) networks involving these marker genes were constructed. RESULTS: The machine learning algorithms identified three genes i.e., SOCS1 (suppressor of cytokine signalling1), MYCN (N-myc proto-oncogene protein), and KLF2 (Kruppel-like factor 2) as diagnostic feature biomarkers associated with ferroptosis. Additionally, CIBERSORT analysis revealed that alterations in the immune microenvironment, such as Macrophages M1, Monocytes, and T cells CD4 naive, could be linked to SOCS1, MYCN, and KLF2. Moreover, the competing endogenous RNA (ceRNA) network exposed a complex regulatory relationship based on marker genes. CONCLUSION: SOCS1, MYCN, and KLF2 are potential biomarkers associated with ferroptosis in SONFH, pending confirmation in future studies. KEY WORDS: Steroid-induced osteonecrosis of the femoral head, Ferroptosis, Machine learning, Genetic analysis.

YTHDF2-Mediated m6A methylation inhibition by miR27a as a protective mechanism against hormonal osteonecrosis in BMSCs.

BACKGROUND: With the increasing incidence of steroid-induced necrosis of the femoral head (SNFH), numerous scholars have investigated its pathogenesis. Current evidence suggests that the imbalance between lipogenesis and osteoblast differentiation in bone marrow mesenchymal stem cells (BMSCs) is a key pathological feature of SNFH. MicroRNAs (miRNAs) have strong gene regulatory effects and can influence the direction of cell differentiation. N6-methyladenosine (m6A) is a prevalent epigenetic modification involved in diverse pathophysiological processes. However, knowledge of how miRNAs regulate m6A-related factors that affect BMSC differentiation is limited. OBJECTIVE: We aimed to investigate the role of miR27a in regulating the expression of YTHDF2 in BMSCs. METHODS: We compared miR27a, YTHDF2, and total m6A mRNA levels in SNFH-affected and control BMSCs. CCK-8 and TUNEL assays were used to assess BMSC proliferation and apoptosis. Western blotting and qRT‒PCR were used to measure the expression of osteogenic (ALP, RUNX2, and OCN) and lipogenic (PPARγ and C/EBPα) markers. Alizarin Red and Oil Red O staining were used to quantify osteogenic and lipogenic differentiation, respectively. miR27a was knocked down or overexpressed to evaluate its impact on BMSC differentiation and its relationship with YTHDF2. Bioinformatics analyses identified YTHDF2 as a differentially expressed gene in SNFH (ROC analysis) and revealed potential signaling pathways through GSEA. The effects of YTHDF2 silencing on the lipogenic and osteogenic functions of BMSCs were assessed. RESULTS: miR27a downregulation and YTHDF2 upregulation were observed in the SNFH BMSCs. miR27a knockdown/overexpression modulated YTHDF2 expression, impacting BMSC differentiation. miR27a silencing decreased m6A methylation and promoted osteogenic differentiation, while YTHDF2 silencing exerted similar effects. GSEA suggested potential signaling pathways associated with YTHDF2 in SNFH. CONCLUSION: miR27a regulates BMSC differentiation through YTHDF2, affecting m6A methylation and promoting osteogenesis. This finding suggests a potential therapeutic target for SNFH.

m6A methylation modification and immune infiltration analysis in osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH) is a elaborate hip disease characterized by collapse of femoral head and osteoarthritis. RNA N6-methyladenosine (m6A) plays a crucial role in a lot of biological processes within eukaryotic cells. However, the role of m6A in the regulation of ONFH remains unclear. In this study, we identified the m6A regulators in ONFH and performed subtype classification. We identified 7 significantly differentially expressed m6A regulators through the analysis of differences between ONFH and normal samples in the Gene Expression Omnibus (GEO) database. A random forest algorithm was employed to monitor these regulators to assess the risk of developing ONFH. We constructed a nomogram based on these 7 regulators. The decision curve analysis suggested that patients can benefit from the nomogram model. We classified the ONFH samples into two m6A models according to these 7 regulators through consensus clustering algorithm. After that, we evaluated those two m6A patterns using principal component analysis. We assessed the scores of those two m6A patterns and their relationship with immune infiltration. We observed a higher m6A score of type A than that of type B. Finally, we performed a cross-validation of crucial m6A regulatory factors in ONFH using external datasets and femoral head bone samples. In conclusion, we believed that the m6A pattern could provide a novel diagnostic strategy and offer new insights for molecularly targeted therapy of ONFH.

Differential Gene Expression and Immune Cell Infiltration in Patients with Steroid-induced Necrosis of the Femoral Head.

OBJECTIVE: The study aimed to study the differential gene expression and immune cell infiltration in patients with steroid-induced necrosis of the femoral head (SANFH), identify the key genes and immune cells of SANFH, and explore the relationship between immune cells and SANFH. METHODS: The high-throughput gene chip dataset GSE123568 was downloaded from the GEO database, and the differential gene expression was analyzed with the R language. The STRING database and Cytoscape software were used to analyze the protein interaction network and screen key genes, and enrichment analysis was carried out on key genes. The infiltration of immune cells in SANFH patients was analyzed and verified by immunohistochemistry. RESULTS: EP300, TRAF6, STAT1, JAK1, CASP8, and JAK2 are key genes in the pathogenesis of SANFH, which mainly involve myeloid cell differentiation, cytokine-mediated signaling pathway, tumor necrosis factor-mediated signaling pathway, and cellular response to tumor necrosis factor through JAK-STAT, NOD-like receptor, toll-like receptor, and other signaling pathways, leading to the occurrence of diseases; immune infiltration and immunohistochemical results have shown the expression of memory B cells and activated dendritic cells as reduced in SANFH patients, while in the same SANFH samples, M1 macrophages have been positively correlated with monocytes, and neutrophils have been negatively correlated with monocytes expression. CONCLUSION: EP300, TRAF6, STAT1, JAK1, CASP8, and JAK2 have exhibited significant differences in SANFH (spontaneous osteonecrosis of the femoral head). Memory B cells, activated dendritic cells, M1 macrophages, monocytes, and neutrophils have shown abnormal expression in SANFH.

Correlation between ESR1 and APOE gene polymorphisms and risk of osteonecrosis of the femoral head: a case-control study.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a disease with a high disability rate, and genetic factors are closely related to its pathogenesis. This study aimed to investigate the possible correlation between ESR1 and APOE gene polymorphisms and the risk of ONFH. METHODS: In this case-control study, the potential association between three genetic variants (rs2982573 C < T, rs10872678 C < T, and rs9322332 A < C) of the ESR1 gene and two genetic variants (rs7259620 A < G and rs769446 C < T) of the APOE gene with the risk of ONFH was investigated. Correlations between gene polymorphisms and ONFH risk were assessed using logistic regression analysis, with calculation of odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: The overall analysis demonstrated that rs9322332 in the ESR1 gene exhibited a correlation with a decreased risk of ONFH under the homozygous (AA vs.CC: OR = 0.69, 95% CI [0.53-0.90], p = 0.006), dominant (CA + AA vs. CC: OR = 0.70, 95% CI [0.54-0.90], p = 0.006), and additive (OR = 0.79, 95% CI [0.66-0.95], p = 0.013) models. The stratification analysis revealed that rs9322332 was linked to a lower risk of ONFH in subgroups characterized by individuals aged over 51 years and non-smokers. Nevertheless, there were no notable correlations found between ESR1 rs2982573 and rs10872678, as well as APOE rs7259620 and rs769446, with the risk of ONFH. CONCLUSION: ESR1-rs9322332 is closely linked to a decreased risk of ONFH, thereby enhancing our understanding of the relationship between gene polymorphisms and ONFH.

Effects of external environment on promoter methylation of PIK3R5 and related pathway regulation in steroid-induced femoral head necrosis.

BACKGROUND: Steroid-induced Avascular Necrosis of the Femoral Head (SANFH) is a condition characterized by the necrosis of the femoral head caused by long-term or high-dose hormone usage. Studies have shown that the PI3K/AKT pathway plays a crucial regulatory role in the development of SANFH. The aim of this study is to determine how external environmental factors induce changes in endogenous hormone levels, how these changes lead to steroid-induced femoral head necrosis, and the interrelationship between the changes in PIK3R5 promoter methylation levels and the regulation of the associated signaling pathways. METHODS: Femoral head samples underwent molecular sequencing analysis. Candidate genes were screened by differential gene analysis and functional enrichment analysis.Methylation level of candidate gene PIK3R5 was verified by methylation-specific PCR(MS-PCR). SANFH model was constructed in New Zealand white rabbits, and the model results were verified by magnetic resonance imaging (MRI) and haematoxylin-eosin (HE) staining.The expression of PIK3R5, PI3K and AKT in rabbit models and human specimens was verified by real-time fluorescence quantitative PCR(RT-qPCR) and Western Blot(WB), respectively. RESULTS: Human femoral head sequencing results indicate distinct differences in the methylation level and mRNA expression of PIK3R5 in SANFH. MS-PCR results showed the methylation level of SANFH patients was significantly higher than that of the control group (P < 0.01). The RT-qPCR results showed that PIK3R5 and PI3K expression levels in the SANFH group were lower than those in the control group (P < 0.05), and the WB experiment results were consistent with the RT-qPCR results. The MRI and HE staining results showed that the rabbit model of SANFH was successfully constructed, and the results of RT-qPCR and WB were consistent with the results of human tissues. CONCLUSION: During the occurrence and development of SANFH, PIK3R5 gene regulates the PI3K/AKT pathway through methylation modification, promotes the oxidative stress response of cells, and accelerates the disease process.

Low miR-182-5p Expressing Extracellular Vesicles Derived From Human Bone Marrow Stromal Cells of Subjects With Steroid-Induced Osteonecrosis of the Femoral Head Aggravate Disease Progression.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory, progressive disease. However, the underlying mechanisms that aggravate femoral head necrosis remain unclear. Extracellular vesicles (EVs) act as molecular carriers in intercellular communication. We hypothesize that EVs derived from human (h) bone marrow stromal cells (BMSC) resident in SONFH lesion areas promote the pathogenesis of SONFH. In the present study, we determined the modulatory effects of SONFH-hBMSCs-derived EVs on the pathogenesis of SONFH in vitro and in vivo. We found that the expression of hsa-miR-182-5p was downregulated in SONFH-hBMSCs and EVs isolated from those hBMSCs. After tail vein injection, EVs isolated from hBMSCs transfected with hsa-miR-182-5p inhibitor aggravated femoral head necrosis in the SONFH mouse model. We conclude that miR-182-5p regulates bone turnover in the SONFH mouse model via targeting MYD88 and subsequent upregulation of RUNX2 expression. We further assume that EVs derived from hBMSCs resident in SONFH lesion areas aggravate femoral head necrosis by downregulating miR-182-5p secreted from hBMSC located outside these lesions. We suggest that miR-182-5p could provide a novel target for future therapeutic approaches to treat or prevent SONFH. © 2023 American Society for Bone and Mineral Research (ASBMR).

Runx2 overexpression promotes bone repair of osteonecrosis of the femoral head (ONFH).

BACKGROUND: Runt-related transcription factor-2 (Runx2) has been considered an inducer to improve bone repair ability of mesenchymal stem cells (MSCs). METHODS AND RESULTS: Twenty-four rabbits were used to establish Osteonecrosis of the femoral head (ONFH) and randomly devided into four groups: Adenovirus Runx2 (Ad-Runx2) group, Runx2-siRNA group, MSCs group and Model group. At 1 week after model establishment, the Ad-Runx2 group was treated with 5 × 107 MSCs transfected through Ad-Runx2, the Runx2-siRNA group was treated with 5 × 107 MSCs transfected through Runx2-siRNA, the MSCs group was injected with 5 × 107 untreated MSCs, and the Model group was treated with saline. The injection was administered at 1 week and 3 weeks after model establishment. The expression of bone morphogenetic protein 2 (BMP-2), Runx2 and Osterix from the femoral head was detected at 3 and 6 weeks after MSCs being injected, and Masson Trichrome Staining, Gross Morphology, X-ray and CT images observation were used to evaluate the repair effect of ONFH. The data revealed that the expression of BMP-2, Runx2 and Osterix in the Runx2-siRNA group was reduced at 3 weeks compared with the MSCs group, and then the expression further reduced at 6 weeks, but was still higher than the Model group besides Osterix; The expression of these three genes in the Ad-Runx2 group was higher than in the MSCs group. Masson Trichrome Staining, Gross Morphology and X-ray and CT images observation revealed that necrotic femoral head of the MSCs group was more regular and smooth than the Runx2-siRNA group, which has a collapsed and irregular femoral head. In the Ad-Runx2 group, necrotic femoral head was basically completely repaired and covered by rich cartilage and bone tissue. CONCLUSIONS: Overexpression of Runx2 can improve osteoblastic phenotype maintenance of MSCs and promote necrotic bone repair of ONFH.

LncAABR07053481 inhibits bone marrow mesenchymal stem cell apoptosis and promotes repair following steroid-induced avascular necrosis.

The osteonecrotic area of steroid-induced avascular necrosis of the femoral head (SANFH) is a hypoxic microenvironment that leads to apoptosis of transplanted bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism remains unclear. Here, we explore the mechanism of hypoxic-induced apoptosis of BMSCs, and use the mechanism to improve the transplantation efficacy of BMSCs. Our results show that the long non-coding RNA AABR07053481 (LncAABR07053481) is downregulated in BMSCs and closely related to the degree of hypoxia. Overexpression of LncAABR07053481 could increase the survival rate of BMSCs. Further exploration of the downstream target gene indicates that LncAABR07053481 acts as a molecular "sponge" of miR-664-2-5p to relieve the silencing effect of miR-664-2-5p on the target gene Notch1. Importantly, the survival rate of BMSCs overexpressing LncAABR07053481 is significantly improved after transplantation, and the repair effect of BMSCs in the osteonecrotic area is also improved. This study reveal the mechanism by which LncAABR07053481 inhibits hypoxia-induced apoptosis of BMSCs by regulating the miR-664-2-5p/Notch1 pathway and its therapeutic effect on SANFH.

Screening and identification of potential key biomarkers for glucocorticoid-induced osteonecrosis of the femoral head.

BACKGROUND: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common disease in osteoarticular surgery, with a high disability rate, which brings great physical and mental pain and economic burden to patients. Its specific pathogenesis has not been fully demonstrated, and there is a lack of recognized effective biomarkers for earlier detection and prompt treatment. This has become an urgent clinical problem for orthopedic scholars. MATERIALS AND METHODS: We downloaded the gene expression profile dataset GSE123568 from the Gene Expression Omnibus database, used STRING and Cytoscape to carry out module analysis and built a gene interaction network. The four core genes most related to GIONFH in this network were ultimately found out by precise analysis and animal experiment were then conducted for verification. In this verification process, thirty-six New Zealand white rabbits were randomly divided into blank control group, model group and drug group. Except for the blank control group, the animal model of GIONFH was established by lipopolysaccharide and methylprednisolone, while the drug group was given the lipid-lowering drugs for intervention as planned. The rabbits were taken for magnetic resonance imaging at different stages, and their femoral head specimens were taken for pathological examination, then the expression of target genes in the femoral head specimens of corresponding groups was detected. Validation methods included RT-PCR and pathological examination. RESULTS: A total of 679 differential genes were selected at first, including 276 up-regulated genes and 403 down-regulated genes. Finally, four genes with the highest degree of correlation were screened. Animal experiment results showed that ASXL1 and BNIP3L were in low expression, while FCGR2A and TYROBP were highly expressed. CONCLUSION: Through animal experiments, it was confirmed that ASXL1, BNIP3L, FCGR2A and TYROBP screened from the comparative analysis of multiple genes in the database were closely related to GIONFH, which is important for early diagnosis of Glucocorticoid-induced osteonecrosis of the femoral head.

[Progress of pathogenesis and genetics of alcohol-induced osteonecrosis of femoral head].

Objective: To review the research progress of pathogenesis and genetics of alcohol-induced osteonecrosis of the femoral head (AIONFH). Methods: The relevant domestic and foreign literature in recent years was extensively reviewed. The pathogenesis, the relationship between gene polymorphism and susceptibility, the related factors of disease progression, and the potential therapeutic targets of AIONFH were summarized. Results: AIONFH is a refractory orthopedic disease caused by excessive drinking, seriously affecting the daily life of patients due to its high disability rate. The pathogenesis of AIONFH includes lipid metabolism disorder, endothelial dysfunction, bone homeostasis imbalance, and et al. Gene polymorphism and non-coding RNA are also involved. The hematological and molecular changes involved in AIONFH may be used as early diagnostic markers and potential therapeutic targets of the disease. Conclusion: The pathogenesis of AIONFH has not been fully elucidated. Research based on genetics, including gene polymorphism and non-coding RNA, combined with next-generation sequencing technology, may provide directions for future research on the mechanism and discovery of potential therapeutic targets.

The analysis of Modified Qing' E Formula on the differential expression of exosomal miRNAs in the femoral head bone tissue of mice with steroid-induced ischemic necrosis of femoral head.

Objective: To investigate the differential expression of exosomal miRNAs in the bone marrow tissue of Modified Qing' E Formula (MQEF) on steroid-induced ischemic necrosis of the femoral head (INFH) model. Methods: Steroid hormones were used to establish the INFH model and treated with MQEF. After successful modeling, femoral tissue exosomes were isolated for miRNA sequencing to obtain femoral tissue exosomal differential miRNAs. By GO analysis and KEGG analysis of the differential genes in both groups, the major exosomal miRNAs of MQEF exerting anti-INFH as well as the major signaling pathways were identified. Next, a quantitative metabolomic validation of MQEF with broad targeting was performed to obtain the main active components of MQEF and to perform biological analysis and signaling pathway prediction of the active components by network pharmacology. Finally, the sequencing results were validated by using RT-qPCR. The results of miRNA sequencing were verified by double examination of network pharmacology and RT-qPCR, and the exosomal miRNAs regulated by the anti-INFH effect of MQEF and the specific signaling pathway of the effect were clarified. Results: A total of 65,389 target genes were predicted in the exosomes of two groups of mice, and 18 significant differentially expressed miRNAs were obtained, of which 14 were up-regulated and 4 down-regulated. GO enrichment analysis showed that these predicted target genes were enriched in 12371 biological processes, 1727 cell components, and 4112 molecular functions. KEGG analysis showed that the predicted miRNA target genes were annotated to 342 signal pathways, in which the highly enriched pathways closely related to bone metabolism were PI3K-Akt signal pathway, MAPK signal pathway, and Wnt signal pathway. The most significantly up-regulated miRNAs were miR-185-3p and miR-1b-5p and the most significantly down-regulated miRNAs were miR-129b-5p and miR-223-5p, of which the targeted genes were closely related to the PI3K-Akt signal pathway. MQEF aqueous decoction extract targeted metabolomics quantitatively combined with network pharmacology predicted targets also closely related to PI3K-Akt signaling pathway. Real-time quantitative PCR validation showed that miR-185-3p was up-regulated 7.2-fold and miR-129b-5p was down-regulated 2.2-fold in the treatment group, and the difference was significant (P < 0.05). Conclusions: MQEF can regulate exosomal miRNA expression in steroid-induced INFH models, miR-185-3p or miR-129b-5p/PI3K-Akt signal axis may be part of the mechanism of MQEF against steroid-induced INFH.

C/EBPα regulates the fate of bone marrow mesenchymal stem cells and steroid-induced avascular necrosis of the femoral head by targeting the PPARγ signalling pathway.

BACKGROUND: The imbalance of osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is closely related to steroid-induced avascular necrosis of the femoral head (SANFH). We aimed to investigate the epigenetic mechanism of intramedullary fat accumulation and continuous osteonecrosis after glucocorticoid (GC) withdrawal in SANFH. METHODS: An SANFH model was established in SD rats, which received an intermittent high GC dose for the first 4 weeks followed by an additional 4 weeks without GC. We explored the synergistic effects and mechanisms of C/EBPα and PPARγ on the differentiation of BMSCs by lentivirus-mediated gene knockdown and overexpression assays. A chromatin immunoprecipitation assay was performed to identify epigenetic modification sites on PPARγ in vivo and in vitro. RESULTS: In the SANFH model, intramedullary fat was significantly increased, and the transcription factors C/EBPα and PPARγ were upregulated simultaneously in the femoral head. In vitro, C/EBPα promoted adipogenic differentiation of BMSCs by targeting the PPARγ signalling pathway, while overexpression of C/EBPα significantly impaired osteogenic differentiation. Further studies demonstrated that histone H3K27 acetylation of PPARγ played an important role in the epigenetic mechanism underlying SANFH. C/EBPα upregulates the histone H3K27 acetylation level in the PPARγ promoter region by inhibiting HDAC1. Additionally, inhibiting the histone acetylation level of PPARγ effectively prevented adipogenic differentiation, thus slowing the progression of SANFH. CONCLUSIONS: Our results demonstrate the molecular mechanism by which C/EBPα regulates PPARγ expression by acetylating histones and revealed the epigenetic phenomenon in SANFH for the first time.

FTO rs62033406 A>G associated with the risk of osteonecrosis of the femoral head among the Chinese Han population.

BACKGROUND: Fat mass and obesity-related (FTO) mRNA was downregulated in osteonecrosis patients. The study aimed to evaluate the correlation between FTO polymorphisms and the susceptibility of osteonecrosis of the femoral head (ONFH). METHODS: Six polymorphisms in FTO were genotyped via the Agena MassARRAY in 498 ONFH patients and 498 healthy controls. Multiple genetic models were used to assess the correlation between FTO polymorphisms and ONFH risk by SNPStats. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using a logistic regression model adjusted by age, gender, smoking and drinking. RESULTS: The risk-increasing association of rs62033406 A>G with ONFH was found (OR = 1.25, 95% CI 1.05-1.50, p = 0.014). Specially, FTO rs62033406 A>G was related to the risk of ONFH in the subgroup at age > 51 years (OR = 1.25, p = 4.00 × 10-4), females (OR = 1.74, p = 1.00 × 10-4), smokers (OR = 1.82, p = 0.005) and drinkers (OR = 1.89, p = 0.002), respectively. The best multi-loci model was the five-loci model, a combination of rs9930333 T>G, rs1558902 T>A, rs56094641 A>G, rs3751812 G>T, and rs62033406 A>G (testing accuracy, 0.5351; p = 0.0004; cross-validation consistency, 10/10). CONCLUSION: Our study first revealed that FTO rs62033406 A>G was a risk factor for ONFH among the Chinese Han population, which might provide the new candidate gene for elucidating the pathogenesis of ONFH.

MicroRNA­141 inhibits the differentiation of bone marrow­derived mesenchymal stem cells in steroid­induced osteonecrosis via E2F3.

Osteonecrosis of the femoral head (ONFH) affects the life of patients. MicroRNA­141 (miR­141) has been found associated with proliferation of bone marrow­derived mesenchymal stem cells (BMSCs). E2F transcription factor 3 (E2F3) has been identified as the target of miR­141 to regulate cell proliferation. The aim of the present study was to investigate whether miR­141 and E2F3 were involved in the osteogenic differentiation of BMSCs during ONFH. BMSCs from 4­week­old Sprague­Dawley rats were transduced with miR­141 mimic or inhibitor lentiviruses. Alkaline phosphatase staining was performed to confirm osteogenic differentiation. Reverse transcription­quantitative PCR, luciferase reporter assays and western blot analysis were also used to examine the interaction between E2F3 and miR­141 in BMSCs from the control and ONFH rats. The lentiviral transductions were carried out successfully. The mRNA expression levels of miR­141 in ONFH were upregulated, while those of E2F3 were downregulated compared with the control rat. The luciferase reporter assays indicated that miR­141 could target E2F3. miR­141 knockdown upregulated the mRNA expression levels of E2F3. In addition, osteogenic differentiation of BMSCs was inhibited following miR­141 overexpression, but increased following miR­141 knockdown, as evidenced by the results of the alkaline phosphatase staining and western blot analysis. In conclusion, miR­141 inhibits the osteogenic differentiation of BMSCs in ONFH by targeting E2F3. These two molecules may represent novel candidates to examine in order to investigate the mechanism underlying ONFH.

Identification of potential autophagy-related genes in steroid-induced osteonecrosis of the femoral head via bioinformatics analysis and experimental verification.

PURPOSE: Steroid-induced osteonecrosis of the femoral head (SONFH) is a refractory orthopaedic hip joint disease that occurs in young- and middle-aged people. Previous experimental studies have shown that autophagy might be involved in the pathological process of SONFH, but the pathogenesis of autophagy in SONFH remains unclear. We aimed to identify and validate the key potential autophagy-related genes involved in SONFH to further illustrate the mechanism of autophagy in SONFH through bioinformatics analysis. METHODS: The GSE123568 mRNA expression profile dataset, including 10 non-SONFH (following steroid administration) samples and 30 SONFH samples, was downloaded from the Gene Expression Omnibus (GEO) database. Autophagy-related genes were obtained from the Human Autophagy Database (HADb). The autophagy-related genes involved in SONFH were screened by intersecting the GSE123568 dataset with the set of autophagy genes. The differentially expressed autophagy-related genes involved in SONFH were identified with R software. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially expressed autophagy-related genes involved in SONFH were conducted by using R software. Then, the correlations between the expression levels of the differentially expressed autophagy-related genes involved in SONFH were confirmed with R software. Moreover, the protein-protein interaction (PPI) network was analysed by using the Search Tool for the Retrieval of Interacting Genes (STRING), significant gene cluster modules were identified with the MCODE Cytoscape plugin, and hub genes among the differentially expressed autophagy-related genes involved in SONFH were screened by using the CytoHubba Cytoscape plugin. Finally, the expression levels of the hub genes of the differentially expressed autophagy-related genes involved in SONFH were validated in hip articular cartilage specimens from necrotic femur heads (NFHs) by using the GSE74089 dataset and further verification by qRT-PCR. RESULTS: A total of 34 differentially expressed autophagy-related genes were identified between the peripheral blood samples of SONFH patients and non-SONFH patients based on the defined criteria, including 25 upregulated genes and 9 downregulated genes. The GO and KEGG pathway enrichment analyses revealed that these 34 differentially expressed autophagy-related genes involved in SONFH were particularly enriched in death domain receptors, the FOXO signalling pathway and apoptosis. Correlation analysis revealed significant correlations among the 34 differentially expressed autophagy-related genes involved in SONFH. The PPI results demonstrated that the 34 differentially expressed autophagy-related genes interacted with each other. Ten hub genes were identified by using the MCC algorithms of CytoHubba. The GSE74089 dataset showed that TNFSF10, PTEN and CFLAR were significantly upregulated while BCL2L1 was significantly downregulated in the hip cartilage specimens, which was consistent with the GSE123568 dataset. TNFSF10, PTEN and BCL2L1 were detected with consistent expression by qRT-PCR. CONCLUSIONS: Thirty-four potential autophagy-related genes involved in SONFH were identified via bioinformatics analysis. TNFSF10, PTEN and BCL2L1 might serve as potential drug targets and biomarkers because they regulate autophagy. These results expand the autophagy-related understanding of SONFH and might be useful in the diagnosis and prognosis of SONFH.

An intron SNP rs2069837 in IL-6 is associated with osteonecrosis of the femoral head development.

BACKGROUND: Genetic polymorphisms play a crucial role in the development of osteonecrosis of the femoral head (ONFH). This study mainly explored the association of IL-6 variants and ONFH susceptibility among the Chinese Han population. METHODS: Two variants (rs2069837, and rs13306435) in the IL-6 gene were identified and genotyped from 566 patients with ONFH and 566 healthy controls. The associations between IL-6 polymorphisms and ONFH susceptibility were assessed using odds ratio (OR) and 95% confidence interval (95% CI) via logistic regression. The potential function of these two variants was predicted by the HaploReg online database. RESULTS: The results of the overall analysis revealed that IL-6 rs2069837 was correlated with decreased risk of ONFH among the Chinese Han population (p < 0.05). In stratified analysis, rs2069837 also reduced the susceptibility to ONFH in older people (> 51 years), males, nonsmokers, and nondrinkers (p < 0.05). However, no associations between rs13306435 and ONFH susceptibility were observed (p > 0.05). CONCLUSIONS: To sum up, we suggested that rs2069837 G>A polymorphism in the IL-6 gene was significantly associated with a decreased risk of ONFH among the Chinese Hans. These findings underscored the crucial role of IL-6 rs2069837 in the occurrence of ONFH.

Association of MIR17HG and MIR155HG gene variants with steroid-induced osteonecrosis of the femoral head in the population of northern China.

INTRODUCTION: Steroid-induced osteonecrosis of the femoral head (ONFH) is a disease of the bone. Metabolism and genetic factors are generally considered to play an important role. The purpose of this study was to investigate the relationship between single-nucleotide polymorphisms (SNPs) in MIR17HG and MIR155HG and the risk of steroid-induced ONFH in the population of northern China. METHODS: A total of 199 steroid-induced ONFH patients and 506 healthy controls were recruited for the study. Four SNPs of MIR17HG and seven SNPs of MIR155HG were genotyped by Sequenom MassARRAY. ORs and 95% CIs were used to evaluate the relationship between these SNPs and steroid-induced ONFH. RESULTS: In the codominant model, patients with the MIR17HG SNPs (rs7318578) AA genotype had an increased risk of steroid-induced ONFH (OR = 1.79, p = 0.039); in the recessive model, patients with the MIR17HG SNP (rs7318578) AA genotype had an increased risk of steroid-induced ONFH (OR = 1.78, p = 0.032). Stratified analysis showed that a MIR17HG SNP (rs7318578) and the MIR155HG SNPs (rs77218221, rs11911469, rs34904192 and rs4143370) were closely related to different unornamented phenotypes of steroid-induced ONFH. Analysis of the clinical indicators revealed significant differences in high-density lipoprotein (HDL-C) levels between the ONFH group and the control group (p = 0.005). In the MIR17HG SNP (rs75267932), patients with different genotypes had different levels of triglyceride (TG). The MIR155HG SNPs (rs77699734, rs1893650, and rs34904192) showed differences in triglyceride (TG), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) levels in patients with different genotypes. CONCLUSION: Our results confirm that MIR17HG and MIR155HG gene mutations are associated with steroid-induced ONFH susceptibility in the population of northern China, providing new evidence for the early detection and prevention of ONFH.