Expression data from KSRP overexpression and knockdown lung cancer cell
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE83388 | GEO | 2016/06/16
SECONDARY ACCESSION(S): PRJNA325776
REPOSITORIES: GEO
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Project description:KSRP knock-down and BMP2 treatment produce a largely overlapping reshape of the transcriptome in C2C12 cells. microRNAs (miRNAs) are essential regulators of development, physiology, and evolution with miRNA biogenesis being strictly controlled at multiple levels. Regulatory proteins, such as KH-type splicing regulatory protein (KSRP), modulate rates and timing of the enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Induction of myogenic miRNAs (myomiRs) is essential for muscle differentiation with KSRP phosphorylation being required to convey myogenic signals to enhanced myomiR maturation. Here we show that either KSRP silencing or Bone Morphogenetic Protein (BMP)2-signaling activation in mesenchimal C2C12 cells prevented myogenic differentiation while induced osteoblastic differentiation as revealed by the reshaping of the whole transcriptome analyzed by RNA deep-sequencing. The most striking feature common to both BMP2 signaling activation and KSRP silencing was a blockade of myomiR maturation. Our results demonstrate that phosphorylated SMAD proteins, the transducers of BMP signaling, associate with KSRP and block its interaction with primary-myomiRs. This, in turn, abrogates KSRP-dependent myomiR maturation with the knock-down of SMAD4, 5, and 9 being able to rescue KSRP function. SMAD-induced blockade of KSRP-dependent myomiR maturation, in parallel to the well known SMAD function on gene transcription, inhibits C2C12 cell differentiation into myofibers and contributes to orient cells towards osteoblast lineage. We propose that remodeling of co-regulatory complexes affecting primary-miRNA processing is a mechanism well suited to guide cell fate determination in eukaryotes. Total RNA was prepared from 1. untreated mock-transfected C2C12 cells; 2. BMP2-treated mock-transfected C2C12 cells; 3. untreated shKSRP-transfected C2C12 cells and analyzed by RNA-seq
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Project description:KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3´-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes in-volved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), in-terleukin (IL)-1β and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. On protein level KSRP-/- BMDM displayed higher levels of these cy-tokines after overnight stimulation. Matching results were observed for primary peritoneal mac-rophages of KSRP−/− mice. These showed higher IL-6, Tumor necrosis factor-α (TNF-α), C-X-C mo-tif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes as an important negative regulator of cytokine expression in macrophages
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