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
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:The post-translational modification of lysine acetylation is related to hepatocellular carcinoma (HCC), and understanding this relationship requires detailed knowledge about the acetylated proteome in HCC tissue and about differences in protein acetylation between cirrhosis and liver cancer. Here we characterized the acetylome in three paired sets of cirrhotic tissue and hepatitis B-related HCC tissue. Combining affinity-based enrichment of acetylated peptides with highly sensitive mass spectrometry, we identified 1493 acetylation sites in 777 proteins and quantified 1040 acetylation sites in 587 proteins. We discovered that the histone acetyltransferases p300 and CBP were hyperacetylated in HCC and cirrhosis but not in normal liver tissue. We also identified several proteins acetylated at more than 10 lysines in HCC and cirrhosis. In addition, our results revealed that HCC was associated with up-regulation of acetylation at 353 sites in 237 proteins and down-regulation of acetylation at 209 sites in 122 proteins. We validated our results using western blotting and immunohistochemistry. Validation with an independent set of clinical samples confirmed our initial screening result that acetylation of lysine 120 in histone H2B type 1-C/E/F/G/I and lysine 18 in histone H3.3 was significantly associated with survival of HCC patients. Acetylation of lysine 77 in histone H4 was associated with survival as well as recurrence. Our study provides numerous new leads to examine the role of acetylation in HCC and its potential as a prognostic marker as well as therapeutic target.
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.
Project description:Background: Of Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), 85%-90% of cases develop from liver cirrhosis, and circular RNAs (circRNAs) have important roles in this process; however, differences in serum circRNA expression profiles between patients with HBV-related cirrhosis and those with HCC have not been studied. Methods: Serum RNA was extracted from patients with HCC and cirrhosis (n = 5 per group) and used for microarray analysis of circRNA expression profiles. Bioinformatics analyses, including clustering, differential expression, and construction of a ceRNA network, were performed. Quantitative real-time reverse transcription PCR validation analysis was conducted using samples from patients with HBV-related cirrhosis (n = 88) and HCC (n = 73). Further, statistical analyses were used to analyze the potential function and value of selected circRNAs with expression differing between the HBV-related cirrhosis and HCC groups. Results: Cluster analysis revealed 8 up-regulated and 80 down-regulated circRNAs. Further, qRT-PCR analysis showed that circRNA_0000367 expression was consistent with that detected by microarray experiments, with significantly lower levels in patients with HBV-related HCC than those with HBV-related cirrhosis. CircRNA_0000367 expression levels were also significantly lower in patients with drug-resistant HBV and those with HBV-related cirrhosis with Model for End-Stage Liver Disease score < 10. Further, circRNA_00000367 expression levels were lower in patients with HBV-related cirrhosis who progressed to HCC. Analysis of the lncRNA-miRNA-mRNA ceRNA network identified 39 miRNAs and 24 mRNAs involved in circRNA_00000367 networks; these target genes were involved in various biological processes and signaling pathways. Conclusion: Serum cicrRNA_0000367 is a potential HCC biomarker in patients with HBV-related cirrhosis, where down-regulation of cicrRNA_0000367 in HBV-related cirrhosis may be associated with progression to HCC.