Cold-inducible RNA-binding Protein Expression Research Articles

The Mechanism of CIRP in Regulation of STAT3 Phosphorylation and Bag-1/S Expression Upon UVB Radiation.

Cold-inducible RNA binding protein (CIRP) is a stress-inducible protein, which could be activated by various cellular stresses, such as hypothermia, hypoxia and UV irradiation. Our previous study indicated that UVB radiation (3 mJ cm-2 ) induces CIRP expression, which promotes keratinocytes growth, survival and eventually transformation via activation of STAT3-Bag-1/S signaling cascade. However, the mechanism(s) of CIRP in regulating p-STAT3 activation and Bag-1/S expression have not been fully elucidated. In this study, we demonstrate that repeated exposure of UVB radiation (3 mJ cm-2 ) or overexpression of CIRP could lead to an elevation of the phosphorylation of Janus kinase (JAK) family proteins (JAK2 and JAK3) in HaCaT cells. The increased phosphorylation of the JAKs correlates to an increased phosphorylation of STAT3 (p-STAT3) in the cells; inhibiting JAKs using JAK inhibitor I lead to a reduction of STAT3 phosphorylation and Bag-1/S expression in wild type HaCaT and CIRP stably transfected HaCaT cells with or without UVB exposure. Furthermore, our data indicated that inhibiting the downstream factor of CIRP, NF-κB, using BAY 11-7085 could also decrease the p-STAT3. These results lead us to propose that CIRP mediates the activation of STAT3-Bag-1/S signaling cascade via activating the JAKs and NF-κB signaling pathways.

Expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma and the effect of CIRP downregulation cell proliferation and chemosensitivity to gemcitabine.

The aim of the present study was to investigate the expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma (RCC) and to determine the effects of downregulation of CIRP on cell proliferation and chemosensitivity to gemcitabine. The expression of CIRP was detected by western blot analysis, quantitative polymerase chain reaction and immunohistochemistry (IHC) in 17 RCC and peri-cancerous tissue samples. Subsequently, the RCC 786-0 cell line was selected in order to investigate the function of CIRP using RNA interference (RNAi) technology, which was able to inhibit the expression of CIRP in vitro. Furthermore, the chemosensitivity to gemcitabine of each group [CIRP small interfering RNA (siCIRP), negative control small interfering RNA (siNC) and blank control] was compared. There were marked differences between the RCC and peri-cancerous tissues. IHC demonstrated that the CIRP expression in 13/17 (76.50%) tumor samples was markedly positive compared with that in the peri-cancerous tissues and the most common pathological type was clear cell RCC (92.30%). This observation was further confirmed through western blot analysis of protein expression levels. CIRP downregulation by RNAi in the RCC 786-0 cell line significantly decreased RCC proliferation. Additionally, when RNAi was coupled with gemcitabine treatment, there was a significant increase in apoptosis in the siCIRP group. CIRP was overexpressed in RCC tissues and in the 786-0 cell line. Downregulation of CIRP by siRNA inhibited the proliferation of the 786-0 cell line and enhanced the chemosensitivity of the cells to gemcitabine. Therefore, CIRP downregulation may provide a novel pathway for the treatment of metastatic RCC.

CIRP increases ICAM-1+ phenotype of neutrophils exhibiting elevated iNOS and NETs in sepsis.

Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1+ neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1+ neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found increased expression of CIRP and higher frequencies and numbers of ICAM-1+ neutrophils in the lungs. Conversely, the CIRP-/- mice showed significant inhibition in the frequencies and numbers of ICAM-1+ neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1+ phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1+ neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared, respectively, with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1+ BMDN were significantly higher than those in ICAM-1- BMDN. Following sepsis the ICAM-1+ neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1- neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP-/- mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus, CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1+ neutrophils in the lungs during sepsis.

The Mechanism Enabling Hibernation in Mammals.

Some rodents including squirrels and hamsters undergo hibernation. During hibernation, body temperature drops to only a few degrees above ambient temperature. The suppression of whole-body energy expenditure is associated with regulated, but not passive, reduction of cellular metabolism. The heart retains the ability to beat constantly, although body temperature drops to less than 10°C during hibernation. Cardiac myocytes of hibernating mammals are characterized by reduced Ca2+ entry into the cell membrane and a concomitant enhancement of Ca2+ release from and reuptake by the sarcoplasmic reticulum. These adaptive changes would help in preventing excessive Ca2+ entry and its overload and in maintaining the resting levels of intracellular Ca2+. Adaptive changes in gene expression in the heart prior to hibernation may be indispensable for acquiring cold resistance. In addition, protective effects of cold-shock proteins are thought to have an important role. We recently reported the unique expression pattern of cold-inducible RNA-binding protein (CIRP) in the hearts of hibernating hamsters. The CIRP mRNA is constitutively expressed in the heart of a nonhibernating euthermic hamster with several different forms probably due to alternative splicing. The short product contained the complete open reading frame for full-length CIRP, while the long product had inserted sequences containing a stop codon, suggesting production of a C-terminal deletion isoform of CIRP. In contrast to nonhibernating hamsters, only the short product was found in hibernating animals. Thus, these results indicate that CIRP expression in the hamster heart is regulated at the level of alternative splicing, which would permit a rapid increment of functional CIRP when entering hibernation. We will summarize the current understanding of the cold-resistant property of the heart in hibernating animals.

Recent progress in the research of cold-inducible RNA-binding protein.

Cold-inducible RNA-binding protein (CIRP) is a cold-shock protein which can be induced after exposure to a moderate cold-shock in different species ranging from amphibians to humans. Expression of CIRP can also be regulated by hypoxia, UV radiation, glucose deprivation, heat stress and H2O2, suggesting that CIRP is a general stress-response protein. In response to stress, CIRP can migrate from the nucleus to the cytoplasm and regulate mRNA stability through its binding site on the 3′-UTR of its targeted mRNAs. Through the regulation of its targets, CIRP has been implicated in multiple cellular process such as cell proliferation, cell survival, circadian modulation, telomere maintenance and tumor formation and progression. In addition, CIRP can also exert its functions by directly interacting with intracellular signaling proteins. Moreover, CIRP can be secreted out of cells. Extracellular CIRP functions as a damage-associated molecular pattern to promote inflammatory responses and plays an important role in both acute and chronic inflammatory diseases. Here, we summarize novel findings of CIRP investigation and hope to provide insights into the role of CIRP in cell biology and diseases.

Expression of RNA-binding Motif Protein 3 (RBM3) and Cold-inducible RNA-binding protein (CIRP) Is Associated with Improved Clinical Outcome in Patients with Colon Cancer.

Recent studies have shown a potential role of RNA-binding proteins (RBPs) in a variety of biological pathways, including cancer progression, whilst their expression in various tumor types may be associated with patient prognosis. However, the role of the RBP family members has not been explored in colon cancer and their possible use as prognostic biomarkers is largely unknown. To determine the prognostic role of three RBP genes: insulin-like growth factor-binding protein 2 (IGFBP2), RNA-binding motif protein 3 (RBM3), and cold-inducible RNA-binding protein (CIRP) in colon cancer. We examined the RNA expression of IGFBP2, RBM3, and CIRP in 94 human colon cancer samples along with matched normal tissue samples from each patient using quantitative real-time polymerase chain reaction (qRT-PCR). No significant associations were observed between RNA expression of RBPs and the studied clinical features. The estimated 5-year disease-free survival rate was significantly better for patients with higher expression of RBM3 and CIRP, while patient survival was not significantly correlated to IGFBP2 expression. RBM3 and CIRP may be useful prognostic biomarkers of colon cancer.

Cloning and expression of cold-inducible RNA binding protein in domestic yak (Bos grunniens).

Cold-inducible RNA binding protein (CIRP) is over-expressed during cold and many other stresses, and could regulate the adaptation to hypothermia. In the present investigation, the objective was to determine the expression of CIRP in adult yak heart, liver, spleen, lung, kidney, brain, ovary, testis and skin by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry from mRNA and protein levels. The CIRP open reading frame encoding was cloned from the domestic yak brain. Results of RT-PCR and Western blot showed the high expression level of CIRP in the heart, kidney, brain, testis and skin, and the lower expression level of CIRP in the lung. Immunohistochemical staining showed CIRP was expressed in the nucleus of neuronal cells, spermatogonia, primary spermatocytes and epidermal cells, and in the cytoplasm of the residual tissues. These observations may provide new data to understand and further study the important role of CIRP protein in the plateau adaptation of the domestic yak on long-term evolution.

Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp.

In mammals, body temperature fluctuates diurnally around a mean value of 36°C-37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells' circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide "approach to steady-state" kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression.

Cold-inducible RNA-binding protein mediates cold air inducible airway mucin production through TLR4/NF-κB signaling pathway

Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases and cold air stimulation has been shown to be associated with the severity of these diseases. However, the regulatory mechanisms that mediate excessive mucin production under cold stress remain elusive. Recently, the cold-inducible RNA-binding protein (CIRP) has been shown to be markedly induced after exposure to cold air. In this study, we sought to explore the expression of CIRP within bronchial biopsy specimens, the effect on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in cold air stimulation process. We found that CIRP protein expression was significantly increased in patients with COPD and in mice treated with cold air. Moreover, cold air stimulation induced MUC5AC expression in wild-type mice but not in CIRP−/− mice. In vitro, cold air stress significantly elevated the transcriptional and protein expression levels of MUC5AC in human bronchial epithelial cells. CIRP, toll-like receptor 4 (TLR4) and phosphorylated NF-κB p65 (p-p65) increased significantly in response to cold stress and CIRP siRNA, TLR4 - neutralizing Ab and a specific inhibitor of NF-κB could attenuated cold stress inducible MUC5AC expression. In addition, CIRP siRNA could hindered the expression levels of TLR4 and p-p65 both induced by cold stress. Taken together, these results suggest that airway epithelial cells constitutively express CIRP in vitro and in vivo. CIRP is responsible for cold-inducible MUC5AC expression by activating TLR4/NF-κB signaling pathway.

Cold-inducible RNA binding protein regulates mucin expression induced by cold temperatures in human airway epithelial cells

Mucus overproduction is an important manifestation of chronic airway inflammatory diseases, however, the mechanisms underlying the association between cold air and mucus overproduction remain unknown. We found that the expression of the cold-inducible RNA binding protein (CIRP) was increased in patients with chronic obstructive pulmonary disease (COPD). In the present study, we tested whether CIRP was involved in inflammatory factors and mucin5AC (MUC5AC) expression after cold stimulation and investigated the potential signaling pathways involved in this process. We found that CIRP was highly expressed in the bronchi of COPD patients. The expression of CIRP, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased, and the CIRP was localized in cytoplasm after cold stimulation. MUC5AC mRNA and protein expression levels were elevated in a temperature- and time-dependent manner after cold stimulation and were associated with the phosphorylation of ERK and NF-κB, which reflected their activation. These responses were suppressed by knockdown of CIRP with a specific siRNA or the ERK and NF-κB inhibitors. These results demonstrated that CIRP was expressed in the bronchi of human COPD patients and was involved in inflammatory factors and MUC5AC expression after cold stimulation through the ERK and NF-κB pathways.

Cold-inducible proteins CIRP and RBM3, a unique couple with activities far beyond the cold.

Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are transcriptionally upregulated in response to low temperature. Featuring an RNA-recognition motif (RRM) and an arginine–glycine-rich (RGG) domain, these proteins display many similarities and specific disparities in the regulation of numerous molecular and cellular events. The resistance to serum withdrawal, endoplasmic reticulum stress, or other harsh conditions conferred by RBM3 has led to its reputation as a survival gene. Once CIRP protein is released from cells, it appears to bolster inflammation, contributing to poor prognosis in septic patients. A variety of human tumor specimens have been analyzed for CIRP and RBM3 expression. Surprisingly, RBM3 expression was primarily found to be positively associated with the survival of chemotherapy-treated patients, while CIRP expression was inversely linked to patient survival. In this comprehensive review, we summarize the evolutionary conservation of CIRP and RBM3 across species as well as their molecular interactions, cellular functions, and roles in diverse physiological and pathological processes, including circadian rhythm, inflammation, neural plasticity, stem cell properties, and cancer development.

Therapeutic Hypothermia Enhances Cold-Inducible RNA-Binding Protein Expression and Inhibits Mitochondrial Apoptosis in a Rat Model of Cardiac Arrest.

Therapeutic hypothermia is well known for its protective effect against brain injury after cardiac arrest, but the exact mechanism remains unclear. Cold-inducible RNA-binding protein (CIRP), a member of cold shock protein, enables mammalian cells to withstand decreased temperature by regulating gene translation. However, the role of CIRP in global cerebral ischemia after therapeutic hypothermia has not been clearly elucidated. In the present study, rats resuscitated from 4min of cardiac arrest were separately treated with therapeutic hypothermia (immediately after return of spontaneous circulation (ROSC); targeted temperature at 33°C) and therapeutic normothermia (targeted temperature at 36.8°C) for 6h. The hippocampus was harvested at 0h (baseline), 6h, 12h, 1day, 3days, and 7days after ROSC. The expression of CIRP messenger RNA (mRNA) was assessed by real-time PCR. CIRP and mitochondrial apoptosis-associated proteins were tested by Western blot. The histological changes and neurological function were respectively evaluated by hematoxylin-eosin staining and neurological deficit score (NDS). Compared with baseline, rats resuscitated from cardiac arrest showed increased expression of CIRP, Bax, Caspase 9, and Caspase 3 and decreased expression of Bcl-2 in hippocampus (P < 0.05). However, therapeutic hypothermia after ROSC alleviated the alterations of Bax, Caspase 9, Caspase 3, and Bcl-2, while further increased the hippocampal expression of CIRP mRNA and protein, when compared with the normothermia rats (P < 0.05). In addition, compared with the therapeutic normothermia rats, histopathological damage in CA1 zone and NDS were respectively decreased and increased in the hypothermia rats (P < 0.05). Our findings suggest that 32°C therapeutic hypothermia exerts an important neuroprotective effects by up-regulating CIRP expression and inhibiting mitochondrial apoptosis factor production in the cardiac arrest rat model.

Involvement of Cold Inducible RNA-Binding Protein in Severe Hypoxia-Induced Growth Arrest of Neural Stem Cells In Vitro

Neonatal hypoxia is the leading cause of brain damage with birth complications. Many studies have reported proliferation-promoting effect of mild hypoxia on neural stem cells (NSCs). However, how severe hypoxia influences the behavior of NSCs has been poorly explored. In the present study, we investigated the effects of 5, 3, and 1 % oxygen exposure on NSCs in vitro. MTT, neurosphere assay, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation revealed a quick growth arrest of C17.2 cells and primary NSCs induced by 1 % oxygen exposure. Cell cycle analysis showed that this hypoxia exposure caused a significant increase of cells in G0/G1 phase and decrease of cells in S phase that is associated with decrease of Cyclin D1. Interestingly, the expression of cold inducible RNA-binding protein (CIRBP), a cold responsive gene reacting to multiple cellular stresses, was decreased in parallel with the 1 % oxygen-induced proliferation inhibition. Forced expression of CIRBP under hypoxia could restore the proliferation of NSCs, as showed by EdU incorporation and cell cycle analysis. Furthermore, the expression of Cyclin D1 under hypoxia was also restored by CIRBP overexpression. Taken together, these data suggested a growth-suppressing effect of severe hypoxia on NSCs and, for the first time, revealed a novel role of CIRBP in hypoxia-induced cell cycle arrest, suggesting that modulating CIRBP may be utilized for preventing hypoxia-induced neonatal brain injury.

Cold-inducible RNA-binding protein plays a central role in the pathogenesis of abdominal aortic aneurysm in a murine experimental model

Cold-inducible RNA-binding protein (CIRP) is a recently identified proinflammatory cytokine. We hypothesize that CIRP is involved in the progression of abdominal aortic aneurysms (AAA) and that anti-CIRP treatment could inhibit this progression. We investigated CIRP expression in the sera and aneurysmal tissues of human AAA patients and elastase-induced AAA rats. To further examine the role of CIRP in the development of AAA, anti-CIRP antibody (1mg/kg) or nonimmunized control immunoglobulin (Ig)G (1mg/kg) was injected via the caudal vein in the experimental AAA model. To further investigate the underlying mechanisms, RAW 267.4cells were stimulated with recombinant murine CIRP (rmCIRP). In human AAA tissue, CIRP exhibited a 5.6-fold and 93% increase in mRNA and protein expression, respectively. In a rat AAA model, CIRP was upregulated significantly in a time-dependent manner in the serum and AAA tissue. The anti-CIRP antibody treatment significantly suppressed the dilation of experimental AAA. Simultaneously, inhibition of CIRP significantly attenuated the expression of matrix metalloproteinase (MMP)-2, MMP-9, tumor necrosis factor-α, and monocyte chemoattractant protein-1, and the number of CD68-positive macrophages in the experimental AAA tissue. Invitro, rmCIRP significantly increased MMP-9 messenger RNA expression in a dose-dependent manner by 1.2-fold, 2.9-fold, and 5.5-fold, respectively. Simultaneously, rmCIRP promoted RAW 264.7cell migration, with an approximately 2.7-fold increase in the number of migrated cells. Our findings demonstrate that CIRP mediates experimental AAA development by promoting the inflammatory response and inducing MMP-9 expression, demonstrating its potential as a novel target for inhibiting the progression of AAA.

Effect of hypothermia on expression of cold-inducible RNA-binding protein after cardiopulmonary resuscitation in a rat model of cardiac arrest

Objective To evaluate the effect of hypothermia on the expression of cold-inducible RNA-binding protein (CIRP) after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest. Methods Ninety pathogen-free male Sprague-Dawley rats, weighing 280-320 g, were randomly divided into 3 groups (n=30 each) using a random number table: sham operation group (group Sham), cardiac arrest-CPR group (group CPR), and hypothermia group (group H). Group Sham underwent only cannulation of the artery and vein and tracheal intubation.In group CPR, cardiac arrest was induced by electric stimulation via the esophagus, and then CPR was performed.In group H, the rectal temperature was cooled down to 32-34 ℃ within 15 min by putting rats on the ice bags immediately after recovery of spontaneous circulation (ROSC), and maintained at this level for 6 h, while the rectal temperature was maintained at 36-37 ℃ in the other groups.Neurological deficit score (NDS) was evaluated at 24 and 72 h after ROSC, and the rats were then sacrificed.The hippocampus was removed for examination of the pathological changes of pyramidal cells in the hippocampal CA1 region (using hematoxylin and eosin staining), and for determination of the expression of CIRP mRNA and protein (using fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction or Western blot). Results Compared with group Sham, NDS score was significantly decreased, and the expression of CIRP mRNA and protein was up-regulated at 24 and 72 h after ROSC in CPR and H groups (P<0.05). Compared with group CPR, NDS score was significantly increased at 72 h after ROSC, and the expression of CIRP mRNA and protein was up-regulated at 24 and 72 h after ROSC in group H (P<0.05). Conclusion Hypothermia can effectively reduce cerebral ischemia-reperfusion injury and improve neurological function after CPR, and the mechanism is probably related to up-regulation of CIRP expression in a rat model of cardiac arrest. Key words: Hypothermia, induced; Heart arrest, induced; Cardiopulmonary resuscitation; Carrier proteins; Hippocampus

Changes in expression of cold-inducible RNA binding protein in hippocampus after cardiopulmonary resuscitation in a rat model of cardiac arrest

Objective To evaluate the changes in the expression of cold-inducible RNA binding protein (CIRP) in the hippocampus after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest. Methods Eighty-five male Sprague-Dawley rats, weighing 280–320 g, aged 3 months, were randomly divided into 3 groups using a random number table: control (group C, n=15), sham operation group (group S, n=15) and CPR group (n=55). Cardiac arrest was induced through ventricular fibrillation caused by transoesophageal cardiac pacing, and then CPR was performed in group CPR.At 6 h, 12 h, and 1, 3 and 7 days after CPR, and the hippocampi were removed for detection of the expression of CIRP mRNA (using real-time reverse transcriptase polymerase chain reaction) and CIRP protein (using Western blot). The brain tissues were also obtained at 3 days after CPR for detection of CIRP expression in the hippocampal CA1 region by immunohistochemistry. Results Compared with group C, the expression of CIRP mRNA was significantly up-regulated at 1, 3, and 7 days after CPR, and the expression of CIRP was up-regulated at 1 and 3 days after CPR in group CRP (P 0.05). Compared with the values at 3 days after CPB, the expression of CIRP mRNA was significantly down-regulated at each time point after CPR, and the expression of CIRP was down-regulated at 6 h, 12 h, and 7 days after CPR in group CRP (P<0.05). CIRP was mainly expressed in the nucleus of pyramidal cells in the hippocampal CA1 region. Conclusion Up-regulated expression of hippocampal CIRP is one of the endogenous mechanisms that inhibit cell apoptosis in the brain tissues of rats. Key words: Heart arrest, induced; Cardiopulmonary resuscitation; RNA-binding proteins; Hippocampus

Developmental competence of mature yak vitrified–warmed oocytes is enhanced by IGF-I via modulation of CIRP during in vitro maturation

The objective of this study was to investigate whether developmental competence of mature vitrified–warmed yak (Bos grunniens) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified–warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified–warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified–warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified–warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification.

Hibernation-specific alternative splicing of the mRNA encoding cold-inducible RNA-binding protein in the hearts of hamsters

The hearts of hibernating animals are capable of maintaining constant beating despite a decrease in body temperature to less than 10 °C during hibernation, suggesting that the hearts of hibernators are highly tolerant to a cold temperature. In the present study, we examined the expression pattern of cold-inducible RNA-binding protein (CIRP) in the hearts of hibernating hamsters, since CIRP plays important roles in protection of various types of cells against harmful effects of cold temperature. RT-PCR analysis revealed that CIRP mRNA is constitutively expressed in the heart of a non-hibernating euthermic hamster with several different forms probably due to alternative splicing. The short product contained the complete open reading frame for full-length CIRP. On the other hand, the long product had inserted sequences containing a stop codon, suggesting production of a C-terminal deletion isoform of CIRP. In contrast to non-hibernating hamsters, only the short product was amplified in hibernating animals. Induction of artificial hypothermia in non-hibernating hamsters did not completely mimic the splicing patterns observed in hibernating animals, although a partial shift from long form mRNA to short form was observed. Our results indicate that CIRP expression in the hamster heart is regulated at the level of alternative splicing, which would permit a rapid increment of functional CIRP when entering hibernation.

Differential modulation of host genes in the kidney of brown trout Salmo trutta during sporogenesis of Tetracapsuloides bryosalmonae (Myxozoa)

Tetracapsuloides bryosalmonae (Myxozoa) is the causative agent of proliferative kidney disease in various species of salmonids in Europe and North America. In Europe, spores of T. bryosalmonae develop in the kidney of infected brown trout Salmo trutta and are released via urine to infect the freshwater bryozoan Fredericella sultana. The transcriptomes of kidneys of infected and non-infected brown trout were compared by suppressive subtractive hybridization. Differential screening and a subsequent NCBI BLAST analysis of expressed sequence tags revealed 21 transcripts with functions that included cell stress and cell growth, ribonucleoprotein, signal transduction, ion transporter, immune response, hemoglobin and calcium metabolisms. Quantitative real time PCR was used to verify the presence of these selected transcripts in brown trout kidney at sporogonic stages of T. bryosalmonae development. Expression of cold-inducible RNA-binding protein, cyclin-dependent kinase inhibitor 2A, prothymosin alpha, transforming protein RhoA, immunoglobulin light chain and major histocompatibility complex class I were up-regulated significantly in infected brown trout. Expression of both the hemoglobin subunit beta and stanniocalcin precursor were down-regulated significantly in infected brown trout. This study suggests that cell stress and cell growth processes, signal transduction activities, erythropoiesis and calcium homeostasis of the host are modulated during sporogonic stages of parasite development, which may support the sporogenesis of T. bryosalmonae in the kidney of brown trout.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0101-z) contains supplementary material, which is available to authorized users.

Protein overexpression of CIRP and TLR4 in oral squamous cell carcinoma: an immunohistochemical and clinical correlation analysis.

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy. Here, we evaluated the expression of cold-inducible RNA-binding protein (CIRP) and toll-like receptors 4 (TLR4) in OSCC tissues with immunohistochemistry. Using biostatistical methods designed to assess the impact of the expression of CIRP and TLR4 on the prognosis of patients with OSCC and relate that expression to the clinicopathological characteristics of these patients. For the first time, we demonstrated that the expression of CIRP and TLR4 was increased in OSCC and that high levels of CIRP or TLR4 expression were associated with a short survival rate. In addition, we were surprised to find that the levels of expression of CIRP and TLR4 were very similar. The goal of this study was to evaluate whether these two genes may provide clues as to the regulatory mechanisms of OSCC, serve as prognostic markers and establish a new direction for further studies of these biological mechanisms.