A Study On The Molecular Mechanism Of Cold-Inducible RNA-Binding Protein Under Chronic Hypoxia And Cold Stress

Objective We sought to explore the intracellular expression and location of cold inducible RNA-binding protein(CIRP) in targeted population, the rat model of chronic hypoxia and in normal human bronchial epithelial cells(NHBE). The rat and cell model of chronic hypoxia-induced cold-sensitive were made. Thus, we sought to explore the role and mechanism of CIRP in cold-provoked infiltration of inflammatory factors in COPD with chronic hypoxia.Methods(1) The location and expression of CIRP in bronchial epithelial cells from COPD patients, health persons, the rats model of cold-provoked exacerbation of COPD with chronic hypoxia and health rats was examined by real-time PCR, western blotting and immunohistochemistry. The location and expression of CIRP in NHBE cells presence or absence with cold air was detected by real-time PCR, western blotting and immunofluorescence staining as well.(2) The knockdown of CIRP plasmid was constructed and transfected into NHBE cells. For transfection, the knockdown of CIRP was measured by real-time PCR and western blotting. The shRNA was established to identify the function of CIRP in hypoxia and cold temperature-induced infiltration of inflammatory factors. CIPR localization and expression in the transfected cells with the optimal cold temperature and time stimulation were determined by immunofluorescence staining. In whole cell lysates from different stimulus groups, mRNA and protein levels of CIRP were examined using real-time PCR and immunoblot analysis, respectively.(3) To identify the stress granules whether was involved in CIRP-mediated inflammatory response. NHBE cells was co-transfected with eIF2α-siRNA and CIPR shRNA. For transfection, the knockdown of eIF2α was tested by real-time PCR and western blotting as well. The formation of stress granules(mark of eIF2α) in the transfected cells with the optimal cold temperature stimulation were determined by immunofluorescence staining. The level of mRNA and protein for CIRP and inflammatory mediators were determined by means of real-time PCR and ELISA, respectively.(4) To clarify CIRP whether has an influence on the stabilization of the mRNA of cytokines in NHBE cells. The dual luciferase reporter vector containing pGL3-TNF-α 3′UTR, pGL4.75-IL-8 3′UTR or pGL3-IL-6 3′UTR reporter gene were co-transfected with pSVRenilla intoNHBE cells which transfected with CIRP shRNA. The relative luciferase activity of reporter genes was measured by dual luciferase reporter(DLR)assay system after the stimulus of cold temperature and hypoxia. 3’UTR of TNF-α, IL-8 and IL-6 mRNA were labeled with biotin in vitro and RNA pull-down assay was performed. The interaction between CIRP and the3’UTR of cytokine mRNAs was investigated using biotin-streptavidin pull-down assay. To examine whether CIRP could influence the mRNA stability of inflammatory mediators. Cells were treated with cold temperature(15 ℃) and hypoxia(8% O2) for 12 h to induce inflammatory mediators transcription, and then transcription was stopped by Actinomycin D(Act D). The total cellular RNA was isolated at the indicated time after treatment with Act D. Inflammatory mediators mRNAs levels were quantified by real-time PCR and the mRNAs half-life(in hours) were analyzed by Opticon2.02 software.Results(1) The results showed that the immunohistochemical staining intensity of CIRP in lung tissues from patients with COPD was substantially stronger than that in normal epithelium samples. Similarly,CIRP staining was significantly stronger in the airway epithelium from rats co-stimulated with hypoxia and cold temperature compared with that of the normal control group. In NHBE cells, we found CIRP positiveimmunoreactivity mainly located in cytoplasm. Moreover, CIRP gene and protein expression levels were significantly greater in the lung tissue of patients with COPD was significantly greater than that in subjects who had no underlying chronic inflammatory airway disease. Meanwhile, hypoxia and cold temperature also induced inceases in CIRP mRNA and protein in the rat model.(2) We constructed the knockdown of CIRP in NHBE cells by using two specific shRNA against to CIRP. For transfection, the knockdown of CIRP was detected by real-time PCR and western blotting. The shRNA was established to identify the function of CIRP in hypoxia and cold temperature-induced infiltration of inflammatory factors. The level of mRNA and protein for inflammatory factors and CIRP were determined by means of real-time PCR, western blotting and ELISA, respectively.(3) Transfection with CIRP-shRNA, eIF2α-siRNA separately or jointly.We found that the percentage of cells containing granules was significantly decreased by immunofluorescence staining. Moreover, the level of mRNA and protein for e IF2α were also attenuated by CIRP-shRNA or eIF2α-siRNA. However, eIF2α-siRNA failed to attenuate the mRNA and protein expressions of CIRP.(4) To investigate whether CIRP regulates the posttranscriptional level of mediator mRNAa by increasing the mediators promoter activity in NHBE cells. The dual luciferase reporter vector containing pGL3-TNF-α3′UTR, pGL4.75-IL-8 3′UTR or pGL3-IL-6 3′UTR reporter gene were co-transfected with pSVRenilla into NHBE cells which transfected with CIRP-shRNA. The results showed that the folds increase in luciferase activity was observed in cells with hypoxia and cold temperature co-treatment, but luciferase activity was weakened by CIRP-shRNA.Foranother, biotinylated transcripts encompassing motif-containing sequences from the mRNAs indicated the formation of complexes with CIRP. Substantial amounts of CIRP were associated with the 3′UTR of TNF-α, IL-8 or IL-6 mRNA, and little CIRP was detected in the normal NHBE cells. The negative control transcripts encompassing the 3′UTR of GAPDH failed to pull down CIRP because they lacked the CIRP motif.Finally, to examine whether CIRP could influence the mRNA stability of inflammatory mediators. Inflammatory mediators mRNAs levels were quantified by real-time PCR and the mRNAs half-life(in hours) were analyzed by Opticon2.02 software. The results demonstrated that the levels of cytokines mRNA increased significantly after the cells were treated with hypoxia and cold. Strikingly, transfection with CIRP shRNA interrupted this cold-upregulated expression of cytokine mRNA at 6 h. The half-life of these mRNA significantly increased when the cells were co-treated with hypoxia and cold, whereas the decay rate of mRNA of inflammatory cytokines was significantly faster when was CIRP knocked down compared with that of cells transfected with scramble shRNA.Conclusion CIRP mRNA and protein expression in human bronchial cells is augmented in COPD patients and the rat chronic hypoxia-induced cold-sensitive model, meanwhile it is gathered in the cytoplasm that next to the lumen of airway. Cold temperature could increase its expression as well,and promote it located in cytoplasm, indicating that CIRP might take an important role in hypoxia and cold-induced inflammatory response. In addition, we showed that CIRP-mediated the production of inflammatory cytokines induced by cold stress through the posttranscriptional regulation.The identification of CIRP-mediated infiltration of inflammatory mediators induced by hypoxia and cold in patients with COPD might help to improve the therapteuic approaches to mitigate infiltration of inflammatory factors.