Cold-inducible RNA-binding Protein Expression Research Articles

Cold-Inducible RNA-Binding Protein but Not Its Antisense lncRNA Is a Direct Negative Regulator of Angiogenesis In Vitro and In Vivo via Regulation of the 14q32 angiomiRs-microRNA-329-3p and microRNA-495-3p.

Inhibition of the 14q32 microRNAs, miR-329-3p and miR-495-3p, improves post-ischemic neovascularization. Cold-inducible RNA-binding protein (CIRBP) facilitates maturation of these microRNAs. We hypothesized that CIRBP deficiency improves post-ischemic angiogenesis via downregulation of 14q32 microRNA expression. We investigated these regulatory mechanisms both in vitro and in vivo. We induced hindlimb ischemia in Cirp−/− and C57Bl/6-J mice, monitored blood flow recovery with laser Doppler perfusion imaging, and assessed neovascularization via immunohistochemistry. Post-ischemic angiogenesis was enhanced in Cirp−/− mice by 34.3% with no effects on arteriogenesis. In vivo at day 7, miR-329-3p and miR-495-3p expression were downregulated in Cirp−/− mice by 40.6% and 36.2%. In HUVECs, CIRBP expression was upregulated under hypothermia, while miR-329-3p and miR-495-3p expression remained unaffected. siRNA-mediated CIRBP knockdown led to the downregulation of CIRBP-splice-variant-1 (CIRBP-SV1), CIRBP antisense long noncoding RNA (lncRNA-CIRBP-AS1), and miR-495-3p with no effects on the expression of CIRBP-SV2-4 or miR-329-3p. siRNA-mediated CIRBP knockdown improved HUVEC migration and tube formation. SiRNA-mediated lncRNA-CIRBP-AS1 knockdown had similar long-term effects. After short incubation times, however, only CIRBP knockdown affected angiogenesis, indicating that the effects of lncRNA-CIRBP-AS1 knockdown were secondary to CIRBP-SV1 downregulation. CIRBP is a negative regulator of angiogenesis in vitro and in vivo and acts, at least in part, through the regulation of miR-329-3p and miR-495-3p.

Normothermic machine perfusion attenuates hepatic ischaemia-reperfusion injury by inhibiting CIRP-mediated oxidative stress and mitochondrial fission.

Extracellular cold‐inducible RNA‐binding protein (CIRP) is a proinflammatory mediator that aggravates ischaemia‐reperfusion injury (IRI). Normothermic machine perfusion (NMP) could effectively alleviate the IRI of the liver, but the underlying mechanism remains to be explored. We show that human DCD livers secreted a large amount of CIRP during static cold storage (CS), which is released into the circulation after reperfusion. The expression of CIRP was related to postoperative IL‐6 levels and liver function. In a rat model, the CIRP expression was upregulated during warm ischaemia and cold storage. Then, rat DCD livers were preserved using CS, hypothermic oxygenated machine perfusion (HOPE) and NMP. C23, a CIRP inhibitor, was administrated in the HOPE group. Compared with CS, NMP significantly inhibited CIRP expression and decreased oxidative stress by downregulating NADPH oxidase and upregulating UCP2. NMP markedly inhibited the mitochondrial fission‐related proteins Drp‐1 and Fis‐1. Further, NMP increased the mitochondrial biogenesis‐related protein, TFAM. NMP significantly reduced inflammatory reactions and apoptosis after reperfusion, and NMP‐preserved liver tissue had higher bile secretion and ICG metabolism compared to the CS group. Moreover, C23 administration attenuated IRI in the HOPE group. Additionally, HL‐7702 cells were stimulated with rhCIRP and C23. High rhCIRP levels increased oxidative stress and apoptosis. In summary, NMP attenuates the IRI of DCD liver by inhibiting CIRP‐mediated oxidative stress and mitochondrial fission.

Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia.

Objective Cold-inducible RNA-binding protein (CIRBP) has been shown to be involved not only in cooling-induced cellular protection but also as a mediator of sterile inflammation, a critical mechanism of the innate immune response in ischemia/reperfusion (I/R) injury. The role of microglia and its activation in cerebral I/R injury warrants further investigation as both detrimental and regenerative properties have been described. Therefore, we investigated the effects of cooling, specifically viability, activation, and release of damage associated molecular patterns (DAMPs) on oxygen glucose deprivation/reperfusion- (OGD/R-) induced injury in murine BV-2 microglial cells. Methods Murine BV-2 microglial cells were exposed to 2 to 6 h OGD (0.2% O2 in glucose- and serum-free medium) followed by up to 19 h of reperfusion, simulated by restoration of oxygen (21% O2) and nutrients. Cells were maintained at either normothermia (37°C) or cooled to 33.5°C, 1 h after experimental start. Cultured supernatants were harvested after exposure to OGD for analysis of DAMP secretions, including high-mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), and CIRBP, and cytotoxicity was assessed by lactate dehydrogenase releases after exposure to OGD and reperfusion. Intracellular cold-shock proteins CIRBP and RNA-binding motif 3 (RBM3) as well as caspases 9, 8, and 3 were also analyzed via Western blot analysis. Furthermore, inducible nitric oxide synthase (iNOS), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-1α (IL-1α), monocyte chemotactic protein 1 (MCP-1), transforming growth factor β (TGFβ), CIRBP, and RBM3 gene expressions were assessed via reverse transcription polymerase chain reaction, and TNF-α, IL-6, and IL-1β releases into the cultured supernatants were assessed via enzyme-linked immunosorbent assays (ELISA). Results Prolonged exposure to OGD resulted in increased BV-2 necrotic cell death, which was attenuated by cooling. Cooling also significantly induced cold-shock proteins CIRBP and RBM3 gene expressions, with CIRBP expression more rapidly regulated than RBM3 and translatable to significantly increased protein expression. DAMPs including HMGB-1, HSP70, and CIRBP could be detected in cultured supernatants after 6 h of OGD with CIRBP release being significantly attenuated by cooling. Exposure to OGD suppressed cytokine gene expressions of IL-1β, TNF-α, MCP-1, and TGFβ independently of temperature management, whereas cooling led to a significant increase in IL-1α gene expression after 6 h of OGD. In the reperfusion phase, TNF-α and MCP-1 gene expressions were increased, and cooling was associated with significantly lower TGFβ gene expression. Interestingly, cooled Normoxia groups had significant upregulations of microglial activation marker, Iba1, IL-1β, and TNF-α gene expressions. Conclusion BV-2 microglial cells undergo necrotic cell death resulting in DAMP release due to OGD/R-induced injury. Cooling conveyed neuroprotection in OGD/R-injury as observable in increased cell viability as well as induced gene expressions of cold shock proteins. As cooling alone resulted in both upregulation of microglial activation, expression of proinflammatory cytokines, and cold shock protein transcript and protein expression, temperature management might have ambiguous effects in sterile inflammation. However, cooling resulted in a significant decrease of extracellular CIRBP, which has recently been characterized as a novel DAMP and a potent initiator and mediator of inflammation.

Cold-inducible RNA-binding protein regulates cyclin B1 against spermatogenesis arrest caused by heat stress.

Spermatogenesis arrest and spermatogenic cell apoptosis occur in the testes of heat-stressed mice. Although heat stress-induced spermatogenic cell apoptosis is due to the decreased expression of cold-inducible RNA-binding protein (CIRBP), it remains unclear whether spermatogenesis arrest is also affected by CIRBP. Additionally, the specific mechanism by which CIRBP regulates spermatogenic cell apoptosis or inhibits spermatogenesis remains to be elucidated. To investigate the mechanism by which CIRBP contributes to heat stress-induced testicular spermatogenesis arrest. Target mRNAs downstream of CIRBP in testicular tissue of BALB/c mice, exposed or not to heat stress, were sequenced. Sequencing data were subjected to bioinformatics analysis to identify key mRNAs and pathways associated with heat stress-induced spermatogenic damage. The link between CIRBP and its target mRNA Ccnb1 (cyclin B1) was verified by western blotting, flow cytometry, and RNA pulldown assays, and the ability of CIRBP to inhibit germ cell cycle arrest by regulating cyclin B1 expression was investigated in a mouse spermatocyte cell line (GC-2spd). Changes in mRNA expression downstream of CIRBP were mainly associated with the cell cycle and RNA binding, transport and splicing. Cyclin B1 was found to regulate the G2/M transition during the first meiotic division of spermatogenic cells. Further, CIRBP was shown to bind directly to the 3'-untranslated region of Ccnb1 mRNA and was associated with cyclin B1-induced inhibition of spermatogenesis arrest. In conclusion, our results provide strong evidence that CIRBP may exert its key function in heat stress-induced testicular spermatogenic cell injury partly by regulating the expression of Ccnb1, the product of which inhibits spermatogenesis arrest.

CIRBP Knockdown Attenuates Tumourigenesis and Improves the Chemosensitivity of Pancreatic Cancer via the Downregulation of DYRK1B

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies worldwide with very limited treatment options. Cold-inducible RNA binding protein (CIRBP) plays promoting roles in several types of cancers, but its function remains unclear in PDAC. Here, we found that the expression of CIRBP was upregulated in PDAC tumor tissues and was significantly associated with poor prognosis. Knockdown of CIRBP in PANC-1 and SW1990 cells inhibited proliferation, migration and invasion in vitro and suppressed tumor growth in vivo. Moreover, CIRBP knockdown enhanced the antitumour effects of gemcitabine treatment in PANC-1 and SW1990 cells, whereas CIRBP overexpression exerted the opposite effects. Mechanistically, CIRBP promoted PDAC malignancy and chemoresistance via upregulation of dual-specificity tyrosine-Y-phosphorylation regulated kinase 1B (DYRK1B). Indeed, knockdown of CIRBP sensitized pancreatic tumors to gemcitabine treatment by diminishing DYRK1B expression and increasing the ratio of ERK/p38 activity. Our findings suggest that CIRBP overexpression facilitates PDAC progression and gemcitabine resistance by upregulating DYRK1B expression and inhibiting the ERK/p38 signaling pathway, highlighting CIRBP as a potential new therapeutic target for PDAC.

CIRBP promotes ferroptosis by interacting with ELAVL1 and activating ferritinophagy during renal ischaemia-reperfusion injury.

Renal ischaemia‐reperfusion (IR) is a major cause of acute kidney injury (AKI). Cold‐inducible RNA‐binding protein (CIRBP) may contribute to AKI because its deficiency protects against renal IR injury in a mechanism believed to involve ferroptosis. We aimed to investigate whether ferroptosis is associated with CIRBP‐mediated renal damage. The differential expression of CIRBP was examined in tubular epithelial (HK2) cells during hypoxia‐reoxygenation (HR) or in response to erastin, an inducer of ferroptosis. CIRBP expression was increased in response to HR or erastin in HK2 cells but the silencing of CIRBP inhibited HR and erastin‐induced ferroptosis together with ferritinophagy. We discovered an interaction between CIRBP and ELAVL1 using STRING software, which was verified through co‐immunoprecipitation and fluorescence colocalization assays. We found that ELAVL1 is a critical regulator in the activation of ferritinophagy and the promotion of ferroptosis. HR or erastin also induced the expression of ELAVL1. An autophagy inhibitor (hydroxychloroquine) or si‐ELAVL1 transfection reversed CIRBP‐enhanced ferritinophagy activation and ferroptosis in HK2 cells under HR. Injection of anti‐CIRBP antibody into a mouse model of IR inhibited ferroptosis and decreased renal IR injury in vivo. In summary, our results provide evidence that ferritinophagy‐mediated ferroptosis could be responsible for CIRBP‐enhanced renal IR injury.

Emodin Alleviates Severe Acute Pancreatitis-Associated Acute Lung Injury by Inhibiting the Cold-Inducible RNA-Binding Protein (CIRP)-Mediated Activation of the NLRP3/IL-1β/CXCL1 Signaling.

Objective: Severe acute pancreatitis (SAP) can lead to acute lung injury (ALI). This study investigated the therapeutic effect of emodin and its molecular mechanisms in a rat model of SAP-ALI. Methods: Forty male Sprague-Dawley rats were randomly divided into the groups: Control (CON), SAP (SAP), emodin (EMO), and C23 (C23). The latter three groups of rats were induced for SAP-ALI by retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct and were treated with vehicle, emodin or C23, respectively. One day post induction, their pancreatic and lung injury was assessed by histology and arterial blood gas analysis. In vitro, rat alveolar macrophages (NR8383 cells) were treated with recombinant rat CIRP in the presence or absence of TAK242 (a TLR4 inhibitor), C23 or emodin. The CIRP-mediated activation of the NLRP3/IL-1β/CXCL1 signaling in rat lungs and NR8383 cells was determined. Similarly, the role of IL-1β in the CIRP-induced CXCL1 expression was investigated. Results: Emodin treatment significantly reduced inflammation and tissue damages in the pancreatic and lung tissues in rats with SAP-ALI, accompanied by decreasing serum amylase, CIRP and IL-1β levels and improving lung function. Furthermore, emodin significantly mitigated the SAP-up-regulated CIRP expression in the pancreatic islets and lung tissues, and attenuated the SAP-activated NF-κB signaling, NLRP3 inflammasome formation and CXCL1 expression in lung resident macrophages as well as neutrophil infiltration in the lungs of rats. In addition, treatment with CIRP significantly activated the NF-κB signaling and NLRP3 inflammasome formation and induced IL-1β and CXCL1 expression and pyroptosis in NR8383 cells, which were abrogated by TAK242 and significantly mitigated by C23 or emodin. Moreover, CIRP only induced very lower levels of CXCL1 expression in IL-1β-silencing NR8383 cells and treatment with IL-1β induced CXCL1 expression in NR8383 cells in a dose and time-dependent manner. Conclusion: Emodin may inhibit the CIRP-activated NLRP3/IL-1β/CXCL1signaling to decrease neutrophil infiltration and ameliorate the SAP-ALI in rats.

What's New in Shock, February 2021?

What's New in Shock, February 2021?

BIOM-03. INVASIVE HISTOPATHOLOGY DRIVES POOR OUTCOMES IN SURGICALLY RESECTED BRAIN METASTASES

Abstract BACKGROUND Surgery as a single modality for the treatment of brain metastases (BrM) results in local recurrence (LR) in 60% of patients. These failure rates are reduced by half with post-operative radiotherapy. The non-invasive nature of BrM has led to the assumption that local recurrence is caused by spillage of cancer cells into the surgical cavity at the time of surgery. We present evidence suggesting that invasion of metastatic cancer cells into the adjacent brain is present in the majority of BrM and is associated with LR, leptomeningeal metastasis (LM), and overall survival (OS). METHODS We assessed the histopathological growth pattern (HGP) of 164 surgically resected BrM. HGP was correlated with LR, LM and OS. Single-cell transcriptomics (scRNAseq) was performed on 15,615 cells from metastasis center (MC) and surrounding brain (SB) adjacent to the tumor. N=30 orthotopic patient-derived xenograft models (OPDX) were established from BrM. RESULTS 56/164 (34%) BrM specimens showed a minimally invasive (MI) HGP between the tumor and adjacent brain while 108/164 (66%) showed significant invasion of tumor lobules or single-cells into the brain (HI-HGP). HI-HGP was associated with LR, LM and shortened OS in BrM patients. scRNAseq identified abundant cancer cells in SB that overexpressed pathways and genes involved in cell survival and stress adaptation compared to matched cancer cells in MC. Validation of these targets with immunohistochemistry in patient and OPDX tissues revealed cold-inducible RNA binding protein (CIRBP) overexpression in HI-HGP patient and OPDX BrM. Modulation of CIRBP expression in OPDX and cell line models of HI-HGP BrM delayed BrM progression and extended OS. CONCLUSION HI-HGP is a poor prognostic indicator in patients with surgically resected BrM, establishing HGP as an important prognostic factor that should be considered by clinicians treating BrM patients. We identify CIRBP as a functional mediator of this process.

65. INVASIVE HISTOPATHOLOGY DRIVES POOR OUTCOMES IN SURGICALLY RESECTED BRAIN METASTASES

BACKGROUNDBrain metastasis (BrM) patients treated with surgery and radiotherapy frequently experience local recurrence (LR), leptomeningeal metastasis (LM), and poor overall survival (OS). We sought to correlate the presence of invasive or circumscribed histopathological growth pattern, observed in the BrM lesion and surrounding brain, with these outcomes, and to study molecular mediators of parenchymal invasion.METHODSWe assessed the HGP of H&E-stained slides from 164 surgically resected BrM from 147 patients. HGP was correlated with incidence of LR, LM and OS. Single-cell RNA sequencing (scRNAseq) was performed on three invasive HGP patients, sampling the metastasis center (MC) and surrounding brain (SB) outside of the contrast-enhancing region. Orthotopic patient-derived xenograft models (OPDX) were established from N=30 brain metastasis via intracranial propagation.RESULTS56/164 BrM specimens (34%) showed a circumscribed growth pattern between the tumor and adjacent brain (cHGP) while 108/164 (66%) showed significant invasion of tumor lobules or single cells into the brain parenchyma (iHGP). iHGP was associated with LR, LM and shortened OS in BrM patients. OPDX models of BrM retain features of patient BrM, including HGP. scRNAseq identified abundant cancer cells in SB that overexpressed a number of genes involved in cell survival, invasion and metastasis compared to matched cancer cells in MC. Validation of these targets with immunohistochemistry in patient and OPDX tissues revealed cold-inducible RNA binding protein (CIRBP) overexpression in iHGP patient and OPDX BrM. Modulation of CIRBP expression in OPDX and cell line models of iHGP BrM delayed BrM progression and extended OS.CONCLUSIONiHGP is a poor prognostic indicator in patients with surgically resected BrM, establishing HGP as an important prognostic factor that should be considered by clinicians treating BrM patients. We identify CIRBP as a functional mediator of this process.

Cold-inducible RNA binding protein agonist enhances the cardioprotective effect of UW solution during extended heart preservation.

In heart transplantation, time restriction is an unavoidable thorny problem during cardiac transport. Cold storage is an important organ preservation method in donor heart transport. Cold-inducible RNA binding protein (CIRBP) has been proven to play a protective role under cold stress. In this study, we investigated the role of CIRBP in hypothermic cardioprotection during heart preservation in UW solution and explored a new approach to extend the heart preservation time. Cirbp-knockout (Cirbp-/- ), Cirbp-transgenic (Cirbp-Tg), and wild-type rats were, respectively, randomized into two groups based on various heart preservation times (6 or 12-hour group) (n=8 per group). After preservation in UW solution, all hearts were mounted on aLangendorff apparatus andunderwent measurement of cardiac parameters, histological analysis, and molecular study. Within the 6-hour preservation group, no significant difference was found in cardiac functions and histological changes between different rat species. However, after 12 hours of preservation, Cirbp-/- rat hearts showed more apoptosis and worse cardiac function, but less apoptosis and better cardiac function were observed in Cirbp-Tg rat hearts. Furthermore, we found CIRBP-mediated cardiac ubiquinone (CoQ10 ) biosynthesis plays an important role in extending heart preservation, and ubiquinone biosynthesis protein COQ9 was an essential down-stream regulator during this process. Finally, we found that zr17-2, a CIRBP agonist, could enhance the expression of CIRBP, which further enhances the synthesis of CoQ10 and promotes scavenging of reactive oxygen species and ATP production to extend heart preservation. This study demonstrated that CIRBP-enhanced CoQ10 biosynthesis during hypothermic heart preservation and zr17-2-supplemented UW solution could be a promising approach to ameliorate heart damage and extend heart preservation during cardiac transport.

Cold-inducible RNA-binding protein contributes to tissue remodeling in chronic rhinosinusitis with nasal polyps.

Cold-inducible RNA-binding protein (CIRP) is a newly identified damage-associated molecular pattern molecule. Its roles beyond promoting inflammation and in human diseases are poorly understood. This study aimed to investigate the involvement of CIRP in chronic rhinosinusitis with nasal polyps (CRSwNP). Immunohistochemistry, quantitative RT-PCR, and ELISA were used to detect the expression of CIRP and matrix metalloproteinases (MMPs) in sinonasal mucosal samples and nasal secretions. Human nasal epithelial cells (HNECs) and THP-1 cells, a human monocytic/macrophage cell line, were cultured to explore the regulation of CIRP expression and MMP expression. Cytoplasmic CIRP expression in nasal epithelial cells and CD68+ macrophages in sinonasal tissues, and CIRP levels in nasal secretions were significantly increased in both patients with eosinophilic and noneosinophilic CRSwNP as compared to those in control subjects. IL-4, IL-13, IL-10, IL-17A, TNF-α, Dermatophagoides pteronyssinus group 1, and lipopolysaccharide induced the production and secretion of CIRP from HNECs and macrophages differentiated from THP-1 cells. CIRP promoted MMP2, MMP7, MMP9, MMP12, and vascular endothelial growth factor A (VEGF-A) production from HNECs, macrophages differentiated from THP-1 cells, and polyp tissues, which was inhibited by the blocking antibody for Toll-like receptor 4, but not advanced glycation end products. The expression of MMPs and VEGF-A in tissues correlated with CIRP levels in nasal secretions in patients with CRSwNP. The upregulated production and release of CIRP from nasal epithelial cells and macrophages may contribute to the edema formation in both eosinophilic and noneosinophilic CRSwNP by inducing MMP and VEGF-A production from epithelial cells and macrophages.

Induction of CIRBP expression by cold shock on bovine cumulus-oocyte complexes.

The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24hr of in vitro maturation of control (38.5°C) and cold-stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.

Hypothermia induces changes in the alternative splicing pattern of cold-inducible RNA-binding protein transcripts in a non-hibernator, the mouse.

Cold-inducible RNA-binding protein (CIRBP) plays important roles in protection against harmful effects of cold temperature. We previously found that several splicing variants of CIRBP mRNA are constitutively expressed in the heart of non-hibernating euthermic hamsters and that one of the variants is predominantly expressed with remarkable reduction in the expression of other variants in hibernating hypothermic hamsters. The aim of this study was to determine whether the regulation of alternative splicing is a common function in a non-hibernator, the mouse. The expression of CIRBP mRNA was assessed by RT-PCR. In euthermic control mice, several splicing variants of CIRBP mRNA were detected in various organs. When hypothermia was induced in mice by using isoflurane anesthesia, the short form variant, which encodes full-length functional CIRBP, was predominantly detected. Keeping body temperature of anesthetized mice at 37°C prevented changes in the splicing pattern. Exposure of mice to a low temperature did not change the splicing pattern, suggesting that endogenous neuronal and/or humoral pathways activated in response to cold stimuli applied to the body surface play minor roles. In agreement with this, the shift in alternative splicing was reproduced in isolated leukocytes in vitro when they were incubated at 28°C. Since application of a TRPM8 or TRPA1 agonist at 37°C failed to promote the shift in the splicing pattern, it seems likely that cold-sensitive channels are not involved in the splicing regulation. Therefore, it is probable that a substantial reduction of temperature is a major cause of the regulation of alternative splicing of CIRBP transcripts. The regulatory system of CIRBP expression at the level of alternative splicing, which was originally discovered in the hibernating hamster, commonly exists in non-hibernators such as mice.

Association between CIRP expression and hypoxic-ischemic brain injury in neonatal rats.

The role of cold inducible RNA-binding protein (CIRP) in mediating ischemic brain injury in neonatal rats under chronic hypobaric hypoxia was investigated. The neonatal rat model of chronic hypobaric hypoxia and the cell culture model of SH-SY5Y cells exposed to hypoxia (1% O2) were constructed. The expression of CIRP and hypoxia-inducible factor-1α (HIF-1α) was detected after hypoxic exposure, and the apoptosis-related proteins were analyzed via terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and western blot analysis to detect neuronal apoptosis. Moreover, the effects of CIRP overexpression on HIF-1α and neuronal apoptosis were identified. Chronic hypobaric hypoxia can lead to HIF-1α expression and neuronal apoptosis in the body. CIRP was induced at early exposure (3 d/7 d). However, the CIRP level in the hypoxic group was obviously lower than that in the control group with the prolongation of exposure time (21 d). In addition, the knockdown of HIF-1α significantly reduced the neuronal apoptosis under hypoxic conditions, indicating that HIF-1α may promote apoptosis during exposure. The overexpression of CIRP significantly inhibited the upregulation of HIF-1α during hypoxia and the HIF-1α-mediated neuronal apoptosis. Results of the current study showed that, CIRP is involved in the ischemic brain injury induced by chronic hypoxia through downregulation of HIF-1α expression.

Cold-inducible RNA-binding protein maintains intestinal barrier during deep hypothermic circulatory arrest.

The intestinal injury during deep hypothermic circulatory arrest (DHCA) is harmful to clinical outcomes. Cold-inducible RNA-binding protein (CIRBP) plays a protective role in hypothermia. The aim of this study was to explore the effects of CIRBP on intestinal barrier during DHCA. Sprague-Dawley (wild type, n = 13) and knockout of Cirbp (Cirbp-/-, n = 8) rats were used in the model of DHCA. The histomorphology of the epithelial barrier was evaluated by haematoxylin-eosin, Chiu's scores, Gram's stain and Ki67. The function of the intestinal barrier was evaluated by serum intestinal fatty acid-binding protein, diamine oxidase and d-lactate. The structure of the epithelial barrier, phosphocreatine-creatine kinase system and adenosine triphosphate were assessed in the intestine. The expression of CIRBP significantly increased in the intestine during DHCA. Cirbp-/- rats showed obvious destruction of intestinal barrier after DHCA. Chiu's scores, intestinal fatty acid-binding protein, diamine oxidase and d-lactate significantly increased in the Cirbp-/- group. Ki67 showed that cell proliferation decreased in the Cirbp-/- rats. In the Cirbp-/- group, zonula occludens-1, E-cadherin and occludin levels were significantly decreased, and these proteins either disappeared or redistributed in the monolayer. Besides, Cirbp-/- resulted in decreased levels of creatine kinase B, glycine amidinotransferase, adenosine triphosphate and creatine contents in the intestine, affecting energy metabolism and balance, which is associated with the maintenance of epithelial barrier during acute injury. CIRBP is related to the maintenance of the intestinal epithelial barrier during DHCA, which is expected to be a new target for the prevention of intestinal injury.

Cold‐Inducible RNA Binding Protein (CIRP) Decreases Inflammation and Breast Tumor Growth

RNA binding proteins (RBPs) post‐transcriptionally regulate gene expression by associating with the regulatory sequences in the untranslated regions of mRNAs. RBPs are often disrupted in cancer, but current knowledge of their role in regulating cancer‐associated genes is largely based on in vitro studies. Cold‐inducible RBP (CIRP) is a stress‐induced RBP that was recently shown to modulate inflammation in response to cellular stress. CIRP expression is altered in several cancers, including breast cancer. The effect of CIRP on inflammation in breast cancer is not known. This study tested the hypothesis, based on our preliminary data and published studies that CIRP changes the inflammatory profile of breast tumors from pro‐ to anti‐tumorigenic, and that CIRP expression and this altered inflammatory milieu are associated with lower tumorgenicity. To test this hypothesis a transgenic mouse overexpressing CIRP in the mammary epithelium was crossed with the PyMT mouse model of breast cancer. Effects on both early (7‐week‐old mice) and late (14‐week‐old mice) tumorigenesis and inflammation were assessed in CIRP/PyMT and PyMT mice. Mammary glands from 7‐week‐old CIRP/PyMT mice had 50% fewer mammary intraepithelial neoplasias than PyMT mice while 14 week‐old CIRP/PyMT mice had slower tumor growth kinetics, smaller tumors, decreased tumor burden and decreased pulmonary metastasis compared to PyMT mice. Importantly, CIRP protein level correlated with decreased tumorigenesis in CIRP/PyMT mice. To assess effects on the inflammatory environment, mammary glands from 7 and 14‐week‐old mice were incubated in medium overnight and Luminex cytokine assays were performed on the medium. Luminex assays were also performed on sera from 14‐wk mice to assess effects on systemic inflammation. There was a decrease in cytokines that promote inflammation, angiogenesis and metastasis at both timepoints for mammary glands, and in 14‐week sera. Overall, these data support a role for CIRP as a potent anti‐tumor molecule that suppresses local and systemic pro‐tumorigenic inflammation. To begin to probe whether CIRP's effects on cytokine levels were cell‐autonomous, we performed in silico analysis of CIRPs binding affinity for individual targets using the web‐based beRBP tool (Yu et al.). CIRP was not predicted to bind the cytokine mRNAs so may be acting in a non‐cell autonomous fashion.Support or Funding InformationNational Institutes of Health Grant/Award Number: F31‐CA213933American Association of Anatomists, Fellows Grant Award ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CIRP regulates BEV-induced cell migration in gliomas.

PurposeA better understanding of the underlying molecular mechanisms in treatment failure of bevacizumab (BEV) for malignant glioma would contribute to overcome therapeutic resistance.MethodsHere, we used a quantitative proteomic method to identify molecular signatures of glioblastoma cell after BEV treatment by two-dimensional liquid chromatography-tandem mass spectrometry analysis and 6-plex iTRAQ quantification. Next, the function of cold-inducible RNA-binding protein (CIRP), one of the most significantly affected proteins by drug treatment, was evaluated in drug resistance of glioma cells by invasion assays and animal xenograft assays. Target molecules bound by CIRP were determined using RNA-binding protein immunoprecipitation and microarray analysis. Then, these mRNAs were identified by quantitative real-time PCR.ResultsEighty-seven proteins were identified with significant fold changes. The biological functional analysis indicated that most of the proteins were involved in the process of cellular signal transduction, cell adhesion, and protein transport. The expression of CIRP greatly decreased after BEV treatment, and ectopic expression of CIRP abolished cell migration in BEV-treated glioma cells. In addition, CIRP could bind mRNA of CXCL12 and inhibit BEV-induced increase of CXCL12 in glioma cells.ConclusionThese data suggested that CIRP may take part in BEV-induced migration of gliomas by binding of migration-relative RNAs.

Effects of cold-inducible RNA-binding protein on the proliferation and apoptosis of spermatogenic cells in vitro following heat stress.

Cold-inducible RNA-binding protein (CIRBP) is reduced by scrotal hyperthermia in cryptorchidism, varicocoele and heat treatment, but there is no direct evidence clarifying the relationship between CIRBP and spermatogenesis. The aim of this study was to investigate the expression of CIRBP in GC2-spd cells (a mouse spermatocyte cell line) before and after heat treatment, and to determine the effects of the downregulation or overexpression of CIRBP on spermatocyte cell proliferation and apoptosis. GC2-spd cells overexpressing CIRBP and GC2-spd cells in CIRBP was knocked down were constructed to investigate the function of CIRBP in cell proliferation and apoptosis using a cell counting kit-8 and flow cytometry respectively. In addition, proliferation and apoptosis were evaluated in GC2-spd cells that had been heated for 30 or 60min, and were analysed 12, 24, and 48h after heat treatment. Heat treatment clearly suppressed the proliferation of GC2-spd cells, and upregulation of CIRBP expression in GC2-spd cells promoted cell proliferation and decreased apoptosis before and after heat stress; in contrast, downregulation of CIRBP expression inhibited cell proliferation and increased apoptosis. These findings suggest that CIRBP exerts a protective effect against spermatogenic injury caused by heat stress.

The regulation of CIRBP by transforming growth factor beta during heat shock-induced testicular injury.

Cold-inducible RNA-binding protein (CIRBP) is associated with cell stress. However, its upstream regulatory factors are still largely unknown. This study investigated whether CIRBP expression was regulated by transforming growth factor beta (TGF-β) during the process of heat-induced testicular damage. Ten male adult ICR mice were allocated to heat treatment (scrotal hyperthermia at 43°C for 30min, n=5) and control group (n=5); CIRBP and TGF-β1, TGF-β2, and TGF-β3 expression levels in the testis in mRNA and protein were analyzed. Then, we conducted invivo and invitro studies to investigate the regulatory effects of TGF-β on CIRBP. In the invivo study, male adult ICR mice were subjected to testicular hyperthermia followed by a local testicular injection of TGF-β antagonist (non-selective TGF-β I/II receptor inhibitor, 5μg or 10μg). In the invitro study, GC2-spd cells were cultured under 43°C for 30min or with different TGF-β isoforms (10ng/mL), and CIRBP expression levels in the testis and GC2-spd cells were analyzed 24 and 48h, respectively, after treatment. As a result, heat treatment significantly downregulated the relative CIRBP mRNA and protein expression (p=0.006 and 0.011), and significantly upregulated TGF-β2 and TGF-β3 expression levels (p=0.022 and 0.04, for mRNA, and p=0.001 for both protein levels). Local testicular injection of 10μg TGF-β antagonist significantly attenuated heat-induced histological damage to the testes and CIRBP downregulation (p=0.038). Furthermore, TGF-β2 and TGF-β3 significantly downregulated CIRBP mRNA and protein expression in GC2-spd cells (all p<0.01), exerting a similar effect to heat treatment. Our invivo and invitro experiments demonstrated that heat-induced CIRBP downregulation in the testes was mediated by the upregulation of TGF-β. Further studies are needed to clarify the molecular mechanisms underlying these processes.