title: Cloning, expression, and purification of cold inducible RNA-binding protein and its neuroprotective mechanism of action.
abstract: BACKGROUND
  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.
authors.affiliation: Department of Neurosurgery, Neuromedical Center, First Affiliated Hospital of PLA General Hospital, No. 51, Fucheng Road, Haidian District, Beijing 100039, China.
authors.forename: Jialin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Neurosurgery, Neuromedical Center, First Affiliated Hospital of PLA General Hospital, No. 51, Fucheng Road, Haidian District, Beijing 100039, China.
authors.forename: Jinghui
authors.initials: J
authors.lastname: Xue
authors.affiliation: Department of Neurosurgery, Neuromedical Center, First Affiliated Hospital of PLA General Hospital, No. 51, Fucheng Road, Haidian District, Beijing 100039, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Neurosurgery, Neuromedical Center, First Affiliated Hospital of PLA General Hospital, No. 51, Fucheng Road, Haidian District, Beijing 100039, China.
authors.forename: Shouchun
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Neurosurgery, Neuromedical Center, First Affiliated Hospital of PLA General Hospital, No. 51, Fucheng Road, Haidian District, Beijing 100039, China.
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Laboratory of Genetic Engineering, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, No. 27, Taiping Road, Haidian District, Beijing 100850, China. Electronic address: Xudg@nic.bmi.ac.cn.
authors.forename: Donggang
authors.initials: D
authors.lastname: Xu
authors.affiliation: Laboratory of Genetic Engineering, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, No. 27, Taiping Road, Haidian District, Beijing 100850, China.
authors.forename: Minji
authors.initials: M
authors.lastname: Zou
authors.affiliation: Department of Neurosurgery, Neuromedical Center, First Affiliated Hospital of PLA General Hospital, No. 51, Fucheng Road, Haidian District, Beijing 100039, China. Electronic address: Zhangzw304@sina.com.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Neuro-oncology Surgery, The General Hospital of Chinese People's Armed Police Forces, No. 69, Yongding Road, Haidian District, Beijing 100039, China.
authors.forename: Jinfu
authors.initials: J
authors.lastname: Diao
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D019938:Cyclin D1 / Q000378:metabolism; D004926:Escherichia coli; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015870:Gene Expression; D006861:Hydrogen Peroxide; D051379:Mice; D018696:Neuroprotective Agents / Q000008:administration & dosage* / Q000302:isolation & purification; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019938:Cyclin D1; D048049:Extracellular Signal-Regulated MAP Kinases; D006861:Hydrogen Peroxide; D018696:Neuroprotective Agents; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C530105:Ccnd1 protein, mouse; C106176:Cirbp protein, mouse; C489428:Myc protein, mouse
keywords: Cell cycle arrest; Cold-inducible RNA-binding protein (CIRP); Neuro2a; Neuroprotective