Project description:Long noncoding RNAs (lncRNAs) have been found to regulate the expression of mRNAs with which they share partial complementarity. We sought to identify the mechanism through which the lncRNA OIP5-AS1, which is abundant in the cytoplasm, suppressed cell proliferation. Silencing of OIP5-AS1 in human cervical carcinoma cells revealed the appearance of many aberrant (monopolar, multipolar, misaligned) mitotic spindles. By biotin-oligomer affinity pulldown, proteomic, and bioinformatic analyses, we identified a subset of human cell cycle regulatory proteins encoded by mRNAs that were capable of interacting with OIP5-AS1. Further investigation revealed that GAK mRNA, which encodes a cyclin G-associated kinase important for mitotic progression, was a prominent target of OIP5-AS1. The interaction between these two transcripts led to a reduction in GAK mRNA stability and GAK protein abundance, as determined in cells in which OIP5-AS1 levels were increased or decreased. Importantly, the aberrant mitotic cell division seen after silencing OIP5-AS1 was partly rescued if GAK was simultaneously silenced. These findings indicate that the abnormal mitoses seen after silencing OIP5-AS1 was caused by an untimely rise in GAK levels and suggest that OIP5-AS1 suppresses cell proliferation at least in part by reducing GAK levels
Project description:The goals of this study are to compare differentially expressed RNAs during OIP5-AS1:MIR-7 TSB treated myoblasts and control myoblasts during myogenesis and further investigate functional regulatory network of those RNAs in myogenesis in vitro and in vivo.
Project description:To investigate the potential pathogenic mechanism of glioma-related epilepsy (GRE), we have employed analyzing of the dynamic expression profiles of microRNA/ mRNA/ lncRNA in brain tissues of glioma patients. Brain tissues of 16 patients with GRE and nine patients with glioma without epilepsy (GNE) were collected. The total RNA was dephosphorylated, labeled, and hybridized to the Agilent Human miRNA Microarray, Release 19.0, 8x60K. The cDNA was labeled and hybridized to the Agilent LncRNA+mRNA Human Gene Expression Microarray V3.0, 4x180K. The raw data was extracted from hybridized images using Agilent Feature Extraction, and quantile normalization was performed using the Agilent GeneSpring. We found that three differentially expressed miRNAs (miR-10a-5p, miR-10b-5p, miR-629-3p), six differentially expressed lncRNAs (TTN-AS1, LINC00641, SNHG14, LINC00894, SNHG1, OIP5-AS1), and 49 differentially expressed mRNAs may play a vitally critical role in developing GRE.
Project description:Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is missing. We employed siRNAs to specifically target GNG12-AS1, a lncRNA overlapping the tumour suppressor DIRAS3, transcriptionally or post-transcriptionally. lncRNA transcriptional silencing by siRNA was mediated by Argounate 2 and led to the upregulation of DIRAS3 transcription through switch in RNA polymerase II binding and active histone marks. Conversely, post-transcriptional silencing of GNG12-AS1 had no effect on DIRAS3 expression. Thus, our findings reveal how RNAi machinery can be used to decouple the process and products of lncRNA transcription. The goal of this study was to identify genes regulated by long noncoding RNA GNG12-AS1 in human cells RNA was extracted from human cells (HB2, SUM159) treated with control and GNG12-AS1 siRNAs. The analysis was performed with six biological replicates for each cell line.
Project description:Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is missing. We employed siRNAs to specifically target GNG12-AS1, a lncRNA overlapping the tumour suppressor DIRAS3, transcriptionally or post-transcriptionally. lncRNA transcriptional silencing by siRNA was mediated by Argounate 2 and led to the upregulation of DIRAS3 transcription through switch in RNA polymerase II binding and active histone marks. Conversely, post-transcriptional silencing of GNG12-AS1 had no effect on DIRAS3 expression. Thus, our findings reveal how RNAi machinery can be used to decouple the process and products of lncRNA transcription. The goal of this study was to identify genes regulated by long noncoding RNA GNG12-AS1 in human cells RNA was extracted from human cells (HB2, SUM159) treated with control and GNG12-AS1 siRNAs. The analysis was performed with six biological replicates for each cell line.
Project description:We identified ADIRF-AS1 as a BMAL1-CLOCK regulated circadian lncRNA. Loss of ADIRF-AS1 in U2OS cells altered rhythmicity of clock-controlled genes and expression of genes associated with cell adhesion and the extracellular matrix (ECM) but did not affect neighboring genes in cis. Affinity based enrichment of U2OS ADIRF-AS1-interacting proteins identified all components of the tumor suppressive PBAF (PBRM1/BRG1) complex. Because PBRM1 is a tumor suppressor mutated in 40% of clear cell renal carcinoma (ccRCC) cases, we studied ccRCC 786O cells and also found PBRM1 bound to ADIRF-AS1. Reducing ADIRF-AS1 expression in 786O and A498 ccRCC cells decreased expression of PBAF-suppressed genes, consistent with ADIRF-AS1 acting to antagonize the function of PBAF. Loss of PBRM1, however, rescued PBAF responsive cell cycle genes in ADIRF-AS1 KO 786O ccRCC cells. Importantly, ADIRF-AS1 expression correlates with survival in human ccRCC, particularly in PBRM1 wild-type, but not mutant PBRM1 tumors. In this regard, loss of ADIRF-AS1 did not affect in vitro 786O cell growth, but strikingly eliminated in vivo tumorigenesis, which was partially rescued by concurrent loss of PBRM1. This rescue, however, requires Matrigel, suggesting a PBRM1-independent function of ADIRF-AS1 in regulating the ECM. Collectively, our findings suggest that ADIRF-AS1 functions partly to antagonize the tumor suppressive effect of the PBAF complex and behaves as an unforeseen BMAL1-regulated, oncogenic lncRNA.
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: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:Long non-coding RNAs (lncRNAs) comprise a diverse class of gene expression regulators with emerging roles in many biological processes including cancer. Here we show that the expression of the lncRNA Hedgehog Interacting Protein Antisense 1 (HHIP-AS1) is a hallmark feature of human SHH-driven tumors. Importantly, loss of HHIP-AS1 leads to reduced tumor growth in SHH-driven tumors in vitro and in vivo. Our results demonstrate the power of cross-entity transcriptome-wide comparisons to identify novel epigenetic–regulatory lncRNA circuitries underlying human cancers.