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Cloning, expression, and purification of cold inducible RNA-binding protein and its neuroprotective mechanism of action.

AbstractBACKGROUND:
Cold-inducible RNA-binding protein (CIRP) is induced in response to hypothermia, where it exerts neuroprotective effects. Our preliminary studies revealed that it inhibits H2O2-induced apoptosis in rat neurons. In the current study, we report effective expression and purification approaches for the synthesis of CIRP, and assess its potential protective effects against oxidative stress.
METHODS:
CIRP-encoding was expressed using the prokaryotic expression system pGEX-4T-1, and SP-Sepharose and Sephacryl S-200 columns were used to purify rCIRP. To mimic ischemia/reperfusion injury-associated oxidative stress, neuro2a cells (N2a) were pre-treated with rCIRP for 2h, followed by hydrogen peroxide (H2O2 60 μmol/ml) for 24h. Cell viability was then quantified using an MTT assay. In addition, western blotting was performed to measure the cell cycle related signal transduction pathways.
RESULTS:
N2a cells exhibited decreased viability following H2O2 treatment, whereas rCIRP significantly improved viability following H2O2 treatment. CIRP also accelerated cell cycle progression from S to G2/M phase in cultured mouse neuroblastoma cells. In addition, CIRP increased levels of p-ERK and p-Akt, and also re-activated the cell cycle-related protein cyclin D1 and c-Myc. These results suggest that CIRP activated the Akt and ERK signal transduction pathways in N2a cells.
CONCLUSIONS:
Our findings suggest that CIRP could exert protective effects against oxidative stress, and that it might be a novel neuroprotective agent.
AuthorsJialin Liu, Jinghui Xue, Haitao Zhang, Shouchun Li, Yuxiao Liu, Donggang Xu, Minji Zou, Zhiwen Zhang, Jinfu Diao
JournalBrain research (Brain Res) Vol. 1597 Pg. 189-95 (Feb 09 2015) ISSN: 1872-6240 [Electronic] Netherlands
PMID25498861 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2015 Elsevier B.V. All rights reserved.
Chemical References
  • Ccnd1 protein, mouse
  • Cirbp protein, mouse
  • Myc protein, mouse
  • Neuroprotective Agents
  • Proto-Oncogene Proteins c-myc
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Cyclin D1
  • Hydrogen Peroxide
  • Proto-Oncogene Proteins c-akt
  • Extracellular Signal-Regulated MAP Kinases
Topics
  • Animals
  • Cell Cycle (drug effects, physiology)
  • Cell Hypoxia (drug effects, physiology)
  • Cell Line, Tumor
  • Cell Survival (drug effects, physiology)
  • Cloning, Molecular
  • Cyclin D1 (metabolism)
  • Escherichia coli
  • Extracellular Signal-Regulated MAP Kinases (metabolism)
  • Gene Expression
  • Hydrogen Peroxide
  • Mice
  • Neuroprotective Agents (administration & dosage, isolation & purification)
  • Oxidative Stress (drug effects, physiology)
  • Phosphatidylinositol 3-Kinases (metabolism)
  • Proto-Oncogene Proteins c-akt (metabolism)
  • Proto-Oncogene Proteins c-myc (metabolism)
  • RNA-Binding Proteins (administration & dosage, genetics, isolation & purification, metabolism)
  • Recombinant Proteins (administration & dosage, genetics, isolation & purification, metabolism)

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