Dataset Information

Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

ABSTRACT: Glutathione-S-transferases mu 2 (GSTM2), a kind of important Phase II antioxidant enzyme of eukaryotes, is degraded by nonsense mediated mRNA decay due to a C27T substitution in the fifth exon of pigs. As a reproductive performance-related gene, GSTM2 is involved in embryo implantation, whereas, functional deficiency of GSTM2 induces pre- or post-natal death in piglets potentially. To have some insight into the role of GSTM2 in embryo development, high throughput RNA sequencing is performed using the swine testis cells (ST) with the deletion of GSTM2. Some embryo development-related genes are observed from a total of 242 differentially expressed genes, including STAT1, SRC, IL-8, DUSP family, CCL family and integrin family. GSTM2 affects expression of SRC, OPN, and SLCs. GSTM2 suppresses phosphorylation of STAT1 by binding to STAT1. In addition, as an important transcription factor, STAT1 regulates expression of uterus receptive-related genes including CCLs, IRF9, IFITs, MXs, and OAS. The present study provides evidence to molecular mechanism of GSTM2 modulating embryo development.

SUBMITTER: Lv Y

PROVIDER: S-EPMC5131268 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

Lv Yuqi Y Jin Yi Y Zhou Yongqiang Y Jin Jianjun J Ma Zhenfa Z Ren Zhuqing Z

Scientific reports 20161201

Glutathione-S-transferases mu 2 (GSTM2), a kind of important Phase II antioxidant enzyme of eukaryotes, is degraded by nonsense mediated mRNA decay due to a C27T substitution in the fifth exon of pigs. As a reproductive performance-related gene, GSTM2 is involved in embryo implantation, whereas, functional deficiency of GSTM2 induces pre- or post-natal death in piglets potentially. To have some insight into the role of GSTM2 in embryo development, high throughput RNA sequencing is performed usin ...[more]

PMID: 27905550

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Project description:BACKGROUND: Protein-tyrosine kinases (PTKs) regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. METHODOLOGY: We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. RESULTS AND CONCLUSION: Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile significantly. Our results indicate that the novel peptide chip technology can be used to unravel kinase signaling pathways in vivo.

Project description:"Bmi-1 and Mel-18 are close structural homologues that belong to the polycomb group (PcG) of transcriptional regulators of homeotic gene expression in development. They are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. A number of clinical and experimental observations have also implicated Bmi-1 in tumorigenesis and stem cell maintenance. Bmi-1 overexpression or amplification has been observed in a number of human malignancies, particularly in B-cell lymphomas, medulloblastomas and breast cancer. We report here that shRNA-mediated knock-down of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival and anchorage-independent growth, and suppression of xenograft tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increased clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone did not affect the growth of SK-OV-3 or U2OS cancer cell lines or normal human WI38 fibroblasts. Gene expression analysis of shRNA-expressing DAOY cells has demonstrated a significant overlap in the Bmi-1- and Mel-18-regulated genes and revealed novel gene targets under their control. Taken together, these results suggest that Bmi-1 and Mel-18 might have overlapping functions in human tumorigenesis. Experiment Overall Design: DAOY cells were transduced with the indicated lentiviral shRNA and stable cell lines were generated by selection in puromycin (1 µg/ml). 2 x 105 cells of each stable cell line were plated onto 60 mm dishes in triplicates and harvested 4 days later. Triplicate total RNA samples from shRNA-expressing and mock-transduced DAOY cells were isolated using affinity resin (Qiagen RNeasy Mini Kit, Qiagen AG). RNA Integrity and purity were assessed with the RNA 6000 Nano LabChip system on Bioanalyzer 2100 (Agilent Technologies). Gene array analysis was conducted at the Genomics Factory at Novartis PHARMA AG, Basel, Switzerland using Gene Chip Human Genome 133 2.0 Plus Expression Array (Affymetrix Inc.)."

Dataset Information

Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

Publications

Deep sequencing of transcriptome profiling of GSTM2 knock-down in swine testis cells.

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