Dataset Information

MiR-145 inhibits the differentiation and proliferation of bone marrow stromal mesenchymal stem cells by GABARAPL1 in steroid-induced femoral head necrosis.

ABSTRACT: Steroid-induced osteonecrosis of femoral head (SANFH) involves impaired differentiation of bone marrow mesenchymal stem cells (BMSC), the mechanism of which is regulated by multiple microRNAs. Studies have shown that miR-145 is a key regulatory molecule of BMSC cells, but its mechanism in steroid-induced femur head necrosis remains unclear. The present study mainly explored the specific mechanism of miR-145 involved in SANFH. In this study dexamethasone, a typical glucocorticoid, was used to induce osteogenic differentiation of BMSC cells. Western blot, qPCR, CCK8 and flow cytometry were used to investigate the effects of miR-145 on the proliferation and differentiation of BMSC. The relationship between miR-145 and GABA Type A Receptor Associated Protein Like 1(GABARAPL1) was identified using dual luciferase reports and the effects of the two molecules on BMSC were investigated in vitro. The results showed that miR-145 was up-regulated in SANFH patients, while GABARAPL1 was down-regulated. Inhibition of miR-145 can improve apoptosis and promote proliferation and activation of BMSC. GABARAPL1 is a downstream target gene of miR-145 and is negatively regulated by miR-145. In conclusion, miR-145 regulates the proliferation and differentiation of glucocorticoid-induced BMSC cells through GABARAPL1 and pharmacologically inhibit targeting miR-145 may provide new aspect for the treatment of SANFH.

SUBMITTER: Xu P

PROVIDER: S-EPMC9701430 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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MiR-145 inhibits the differentiation and proliferation of bone marrow stromal mesenchymal stem cells by GABARAPL1 in steroid-induced femoral head necrosis.

Xu Pengfei P Chang Jun J Ma Guangwen G Liao Faxue F Xu Tangbing T Wu Yunfeng Y Yin Zongsheng Z

BMC musculoskeletal disorders 20221126 1

Steroid-induced osteonecrosis of femoral head (SANFH) involves impaired differentiation of bone marrow mesenchymal stem cells (BMSC), the mechanism of which is regulated by multiple microRNAs. Studies have shown that miR-145 is a key regulatory molecule of BMSC cells, but its mechanism in steroid-induced femur head necrosis remains unclear. The present study mainly explored the specific mechanism of miR-145 involved in SANFH. In this study dexamethasone, a typical glucocorticoid, was used to ind ...[more]

PMID: 36435763

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Project description:The steriod-induced necrosis of femoral head(SINFH) is a serious clinical problem and the underlying mechanism of this disease remains unknown. As its etiology and pathogenesis are not clear, there hasn’t been an effective treatment yet. In-depth studies on its pathogenesis will provide important information for the prevention and treatment of this devastated disease. Animal model researches are helpful to a better understanding of its etiology and pathogenesis, to early diagnosis and treatments and provide the basis for clinical treatment It has been proved that the occurrence of SINFH is related to gene itself and its polymorphism. These studies, in which only one or a few genes were investigated, could not provide a comprehensive understanding to the pathogenesis of SINFH. We used microarrays to compare the gene expression profile of SINFH rats with that of normal rats and identified gene expression changes between SINFH rats and normal rats. Adult healthy male Wistar rats(body weight 200±10g) were randomly divided into control and experimental groups. All the rats were given injection of E. coli endotoxin at a dose of 20μg/kg body weight into abdominal cavity for two times with a 24 hour interval. 24 hours after the second administration of E. coli endotoxin, methylprednisolone 40mg/kg was administered into the left gluteus muscle of experimental group rats for three times with a 24 hour interval. Rats were then killed 6 weeks after steroid administration. In experiment, immediately after death the left femoral heads were isolated. Femoral heads were used for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the gene expression profile of SINFH rats and that of normal rats in order to identified gene expression changes between SINFH rats and normal rats by comparing the gene expression profile of SINFH rats with that of normal rats. To that end, we have established the gene expression profiles of normal and SINFH disease of rat femoral head and found out differentially expressed genes.

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.

Dataset Information

MiR-145 inhibits the differentiation and proliferation of bone marrow stromal mesenchymal stem cells by GABARAPL1 in steroid-induced femoral head necrosis.

Publications

MiR-145 inhibits the differentiation and proliferation of bone marrow stromal mesenchymal stem cells by GABARAPL1 in steroid-induced femoral head necrosis.

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