Project description:Steroid-induced avascular necrosis of the femoral head (SANFH) is closely associated with the imbalance between adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Additionally, epigenetic regulation plays a critical role in this process. Our previous research found that during BMSC adipogenic differentiation, C/EBPα enhances the histone H3K27 acetylation modification at the PPARγ promoter, promoting sustained adipogenic differentiation of BMSCs, suggesting that Histone deacetylases (HDACs) may play an important role in BMSC adipogenic differentiation. However, identifying specific HDAC target genes requires further investigation. This study combines cell experiments with clinical specimen experiments to screen specific HDAC genes involved in BMSC adipogenic differentiation and explore their preliminary functions. Our findings indicate that HDAC10 influences the progression of steroid-induced avascular necrosis of the femoral head by regulating BMSC adipogenic differentiation, possibly through its association with PPARγ histone acetylation. These discoveries provide promising directions for the treatment of steroid-induced avascular necrosis of the femoral head.
Project description:Wistar Kyoto Rats were administered glucocorticoid pellets and placebo pellets for 6 months. After 6 months rats were sacrificed and their femoral heads were histologically examined for the detection of avascular necrosis of the femoral head. Total RNA was extracted from femoral heads and submitted to gene chip microarray for differential gene expression analysis..
Project description:The pathogenesis of necrosis of femoral head (NFH) remains elusive now. Limited studies were conducted to investigate the molecular mechanism of hip articular cartilage damage of NFH. We conducted a genome-wide gene expression profiling of hip articular cartilage with NFH.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.