Project description:Glioblastoma multiforme is the most common and aggressive form of brain cancer. The use of oncolytic HSV-1 (oHSV) to selectively target brain cancer cells leading to their lytic destruction has shown to be very promising in a preclinical setting, but is lacking efficacy in clinical trials. Cyr61, a secreted extracellular matrix protein which functions to promote angiogenesis, migration, proliferation and tumorigenesis, was found to be upregulated rapidly following oHSV infection. Here we show, using microarray analysis, that Cyr61 expression leads to the induction of several genes with type 1 interferon function. We show that Cyr61 mediated type 1 IFN induction is through its interaction with integrin alpha6beta1 on the cell surface and results in oHSV inhibition, reducing the efficacy of this therapy. We used microarray to detail the global program of gene expression underlying Cyr61 mediated oncolytic HSV-1 inhibition and identified distinct classes of up-regulated genes during this process. Tetracycline-Inducible glioma cells expressing Cyr61 protein in the presence of doxycycline were treated with or without doxycycline for 24 hours. RNA was extracted and hybridized on Affymetrix microarray. Two groups: ± dox to induce cyr61, performed in triplicate.
Project description:Newcastle disease virus (NDV) has emerged as an oncolytic agent in several cancers. Previous study has shown that NDV exerts cytolytic activity in glioma, however, the underlying mechanism has not been fully uncovered. Here the cytolytic activity of NDV in glioma and the associated mechanisms have been demonstrated. Infection with NDV inhibits cell proliferation and promotes cell apoptosis in LN229 cells. Further investigation showed that cytoplasmic organelle damage and cytoplasmic vacuolation were observed in LN229 cells after NDV infection. JC-1 staining assay proved that NDV caused cell apoptosis of LN229 cells by inducing mitochondrial dysfunction. We next speculated that NDV caused LN229 cells death through inducing necroptosis, but not ferroptosis, since the Fe2+ level did not alter after NDV infection. Furthermore, the NDV-caused cell death in LN229 cells was blocked by necroptosis inhibitor Nec1. Besides, RNA-seq analysis identified the different expression genes in NDV-infected LN229 cells. OASL, an antiviral gene, has been found to be directly induced by NDV infection. We also found that knockdown of OASL enhanced NDV infection-induced LN229 cells necroptosis. In summary, two aspects about cytolytic activity of NDV in glioma have been demonstrated. NDV presented cytolytic activity in glioma cells through inducing necroptosis. Additionally, targeting OASL may provide new strategy for enhancing necroptosis of glioma cells after NDV infection.
Project description:Knockdown of Sox2 in LN229 gliomal cancer cells decrease their growth rates in vitro. We used microarrays to detail the global programme of gene expression in Sox2 Knockdown LN229 cells compared with mock knockdown LN229 cells Sox2 knockdown LN229 cells and and mock knockdown LN229 cells were cultured in DMEM cell culture media for RNA extraction and hybridization on Applied Biosystems Human Genome Survey Microarrays . We sought to obtain the genes regulated by Sox2 in glioma cell line.
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:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.