as a common intracranial benign tumor , pituitary adenoma accounts for about 10% of all intracranial tumors and has an increasing rate of incidence . pituitary adenoma stimulates hypophysis to over - secrete hormones , which can disrupt the body endocrine system , further causing damage to multiple tissues and organs . the tumor - induced pituitary hypo - function can also severely impair patient quality of life , endanger physical and mental heal , or even cause death . although pituitary adenoma is widely accepted as a benign intracranial tumor , it is often found to invade toward peripheral brain tissues , including cavernous sinus on both sides , sphenoid sinus , suprasellar and parasellar regions , or even damage brain tissues and structures such as those in the sellar region and vomer peripheral region . thus pituitary adenoma may have some biological features of malignant tumors , causing the lower rate of complete resection and higher post - op recurrence rate , even when being assisted with radio- or chemo - therapy . therefore , it is extremely difficult to completely cure pituitary adenoma , which is now a major health issue and attracts wide research attention . pituitary adenoma recurrence has been well defined by studies on biological behaviors of pituitary tumors . complicated mechanisms , including multiple steps and various factors related with tumor invasiveness and growth , are involved in the pathogenesis of recurrent pituitary adenoma . current opinions agree that pituitary adenoma s biological behavior is difficult to evaluate objectively solely based on clinical diagnostic methods such as symptoms and radiological and histopathological examinations . therefore , current clinical research focuses on finding an accurate and simple method to detect the tumor type in order to provide theoretical evidence for the diagnosis of recurrent pituitary adenoma . cold - inducible rna - binding protein ( cirp ) was first identified from mouse testicular cells in 1997 and was later proved to be expressed in various cells , including recombinant cells of human and mammalians as more knowledge has been obtained about cirp , it is found to participate in various biological activities beyond the cold - induced response , suggesting its critical role in body physiological functions . changes in temperature , hypoxia , local ischemia , hydrogen peroxide , and osmotic pressures can induce the expression of cirp , which participates in regulating endometrial cycles , embryonic development , oncogenesis , and hypothermia protecting tumor necrotic factor - induced cell apoptosis , in addition to the elevation of recombinant protein yields via its over - expression in recombinant cells [ 1214 ] . the potential role of cirp expression in pituitary adenoma as a tumor s biomarker , however , has not been fully defined . this study therefore aims to measure the expression profile of cirp in pituitary adenoma and to investigate its effects on tumor recurrence . we selected 60 pituitary adenoma patients admitted to our department of neurosurgery , shandong provincial hospital , shandong university between january 2011 and december 2013 . there were 27 males and 33 females included in this study , with age between 24 and 58 years ( average=33.415.3 years ) . in a pathological classification , there were 20 patients with invasive pituitary adenoma , 20 patients with non - invasive pituitary adenoma , and 20 patients with non - invasive recurrent adenoma . all patients presented clinical symptoms consistent with diagnostic criteria and were confirmed by radiology , or surgical and pathological examinations . all included patients had no statistical significant difference in general information such as age or sex . the protocol of this study was pre - approved by the ethics committee of our hospital . tumor samples collected from surgical resection were divided into small cubes in a sterile environment and were stored in cryopreservation tubes , which were immediately stored in liquid nitrogen for further use . chemical reagents in western blotting were purchased from beyotime biotech ( shanghai , china ) . trizol reagents , rna extraction kit , rt - pcr primers , rt kits , and real time - pcr reagents were all purchased from invitrogen ( us ) . rabbit anti - human cirp antibody was a product of dpc biermann ( germany ) . mouse anti - rabbit igg secondary antibody horseradish peroxidase conjugate was purchased from cell signaling ( usa ) . total tissue proteins were extracted from pituitary adenoma . in brief , lysis buffer was added to tissues and kept on ice for 15~30 min , followed by ultrasonic cell rupture for 5 sec  4 times . supernatants were then transferred to a new eppendorf tube , in which proteins were quantified and kept at 20c for western blotting . tissues lysis mixtures after processing were added with loading buffer , denatured at 95c for 5 min , and separated using 10% sds - page protein electrophoresis . after separation , proteins were then transferred onto the pvdf membrane and blocked in defatted milk powder for 2 h at room temperature to decrease non - specific backgrounds . cirp antibody diluted in 1:500 was incubated with the membrane at 4c overnight and washed with pbst . mouse anti - rabbit igg secondary antibody ( 1:2 000 ) was added to incubate the membrane for 30 min at room temperature in the dark . after a general pbst washing , enhanced ecl chromogenic substrate was used to develop the membrane for 1 min , followed by x - ray exposure ; images were scanned by bio - rad image analysis system and density of bands were measured by quantity one software . anti - actin antibody was used an internal reference to measure sample volume of all groups . all experiments were repeated 4 times ( n=4 ) and data were collected and analyzed . in a sterile environment , frozen tissue samples of pituitary adenoma were quickly ground on ice using a tissue - grinding rod . after complete homogenization , tissue mixtures were eluted repeatedly with 150 l buffer to make a clear solution , which was then transferred to sterile centrifuged tubes . using high - speed centrifugation at 20 000 rpm for 2 min , ( forward primer for cirp : 5-caagt acgga cagat ctctg a-3 ; reverse primer for cirp : 5-cggat ctgcc gtcca tcta-3 ; forward primer for gapdh : 5-agtac cagtc tgttg ctgg-3 ; reverse primer for gapdh : 5 taata gaccc ggatg tctgg t-3 ) . real - time pcr used the following condition : 50c for 1 min ; 95c denature 30 s , 56 or 60c annealing for 50 s , plus 72c extension for 35 sec , repeated for 35 cycles . using gapdh as an internal reference , data were collected based on ct numbers calculated from built - in software of the fluorescence quantitative pcr reactor . a standard curve was plotted from ct values of standard samples and acted as a basis for quantitative analysis using the 2 method . spss 12.0 software package ( ibm corp . , usa ) was used to process all collected data . enumeration data were analyzed in chi - square test , while measurement data are presented as mean  standard deviation ( sd ) . we selected 60 pituitary adenoma patients admitted to our department of neurosurgery , shandong provincial hospital , shandong university between january 2011 and december 2013 . there were 27 males and 33 females included in this study , with age between 24 and 58 years ( average=33.415.3 years ) . in a pathological classification , there were 20 patients with invasive pituitary adenoma , 20 patients with non - invasive pituitary adenoma , and 20 patients with non - invasive recurrent adenoma . all patients presented clinical symptoms consistent with diagnostic criteria and were confirmed by radiology , or surgical and pathological examinations . all included patients had no statistical significant difference in general information such as age or sex . the protocol of this study was pre - approved by the ethics committee of our hospital . tumor samples collected from surgical resection were divided into small cubes in a sterile environment and were stored in cryopreservation tubes , which were immediately stored in liquid nitrogen for further use . chemical reagents in western blotting were purchased from beyotime biotech ( shanghai , china ) . trizol reagents , rna extraction kit , rt - pcr primers , rt kits , and real time - pcr reagents were all purchased from invitrogen ( us ) . rabbit anti - human cirp antibody was a product of dpc biermann ( germany ) . mouse anti - rabbit igg secondary antibody horseradish peroxidase conjugate was purchased from cell signaling ( usa ) . total tissue proteins were extracted from pituitary adenoma . in brief , lysis buffer was added to tissues and kept on ice for 15~30 min , followed by ultrasonic cell rupture for 5 sec  4 times . supernatants were then transferred to a new eppendorf tube , in which proteins were quantified and kept at 20c for western blotting . tissues lysis mixtures after processing were added with loading buffer , denatured at 95c for 5 min , and separated using 10% sds - page protein electrophoresis . after separation , proteins were then transferred onto the pvdf membrane and blocked in defatted milk powder for 2 h at room temperature to decrease non - specific backgrounds . cirp antibody diluted in 1:500 was incubated with the membrane at 4c overnight and washed with pbst . mouse anti - rabbit igg secondary antibody ( 1:2 000 ) was added to incubate the membrane for 30 min at room temperature in the dark . after a general pbst washing , enhanced ecl chromogenic substrate was used to develop the membrane for 1 min , followed by x - ray exposure ; images were scanned by bio - rad image analysis system and density of bands were measured by quantity one software . anti - actin antibody was used an internal reference to measure sample volume of all groups . all experiments were repeated 4 times ( n=4 ) and data were collected and analyzed . in a sterile environment , frozen tissue samples of pituitary adenoma were quickly ground on ice using a tissue - grinding rod . after complete homogenization , tissue mixtures were eluted repeatedly with 150 l buffer to make a clear solution , which was then transferred to sterile centrifuged tubes . using high - speed centrifugation at 20 000 rpm for 2 min , ( forward primer for cirp : 5-caagt acgga cagat ctctg a-3 ; reverse primer for cirp : 5-cggat ctgcc gtcca tcta-3 ; forward primer for gapdh : 5-agtac cagtc tgttg ctgg-3 ; reverse primer for gapdh : 5 taata gaccc ggatg tctgg t-3 ) . real - time pcr used the following condition : 50c for 1 min ; 95c denature 30 s , 56 or 60c annealing for 50 s , plus 72c extension for 35 sec , repeated for 35 cycles . using gapdh as an internal reference , data were collected based on ct numbers calculated from built - in software of the fluorescence quantitative pcr reactor . a standard curve was plotted from ct values of standard samples and acted as a basis for quantitative analysis using the 2 method . spss 12.0 software package ( ibm corp . , usa ) was used to process all collected data . enumeration data were analyzed in chi - square test , while measurement data are presented as mean  standard deviation ( sd ) . an analysis of clinical records of pituitary adenoma patients , including invasive adenoma , non - invasive adenoma , and non - invasive recurrent adenoma , showed that the majority of patients in all 3 tumor types were young people , with an average age of 33.415.3 years . patients among all 3 types had no statistically significant differences in age or sex , as shown in table 1 . there was no statistical difference in general information across all included patients ( p>0.05 ) . tissue proteins were extracted from samples of invasive pituitary adenoma , non - invasive adenoma , and non - invasive recurrent adenoma , and were semi - quantitatively analyzed by western blotting . results showed that cirp protein was expressed in all 3 types of pituitary adenoma tissues , but with different levels : non - invasive adenoma had the lowest cirp proteins ; invasive pituitary adenoma had significantly higher cirp protein levels compared to non - invasive tumors with statistical significance ( p<0.05 ) ; and cirp protein expression was further elevated for recurrent pituitary adenoma with statistically significant difference compared to non - recurrent tumors ( p<0.05 ) as shown in figures 1 and 2 . these results suggest that cirp is directly related with tumor oncogenesis and progression , and may be an important biomarker . to further confirm the expressional profile of cirp mrna in pituitary adenoma , mrna was extracted from samples of invasive pituitary adenoma , non - invasive adenoma , and non - invasive recurrent adenoma and was semi - quantitatively analyzed by real - time pcr . results showed that cirp mrna existed in all 3 types of adenoma , but with different levels . non - invasive pituitary adenoma has the lowest cirp mrna level , which was significantly increased in invasive pituitary adenoma ( p<0.05 compared to non - invasive adenoma ) . the highest level of cirp was found in recurrent pituitary adenoma , with a significant difference ( p<0.05 compared to non - recurrent tumors ) ( figure 3 ) . an analysis of clinical records of pituitary adenoma patients , including invasive adenoma , non - invasive adenoma , and non - invasive recurrent adenoma , showed that the majority of patients in all 3 tumor types were young people , with an average age of 33.415.3 years . patients among all 3 types had no statistically significant differences in age or sex , as shown in table 1 . there was no statistical difference in general information across all included patients ( p>0.05 ) . tissue proteins were extracted from samples of invasive pituitary adenoma , non - invasive adenoma , and non - invasive recurrent adenoma , and were semi - quantitatively analyzed by western blotting . results showed that cirp protein was expressed in all 3 types of pituitary adenoma tissues , but with different levels : non - invasive adenoma had the lowest cirp proteins ; invasive pituitary adenoma had significantly higher cirp protein levels compared to non - invasive tumors with statistical significance ( p<0.05 ) ; and cirp protein expression was further elevated for recurrent pituitary adenoma with statistically significant difference compared to non - recurrent tumors ( p<0.05 ) as shown in figures 1 and 2 . these results suggest that cirp is directly related with tumor oncogenesis and progression , and may be an important biomarker . to further confirm the expressional profile of cirp mrna in pituitary adenoma , mrna was extracted from samples of invasive pituitary adenoma , non - invasive adenoma , and non - invasive recurrent adenoma and was semi - quantitatively analyzed by real - time pcr . results showed that cirp mrna existed in all 3 types of adenoma , but with different levels . non - invasive pituitary adenoma has the lowest cirp mrna level , which was significantly increased in invasive pituitary adenoma ( p<0.05 compared to non - invasive adenoma ) . the highest level of cirp was found in recurrent pituitary adenoma , with a significant difference ( p<0.05 compared to non - recurrent tumors ) ( figure 3 ) . derived mainly from monoclonal adenoma of adenohypophysis gland cells , pituitary adenoma is a common intracranial tumor with a high incidence , which shows a younger trend , as most patients are young men between 20 and 50 years of age , with no sex bias . this study randomly selected pituitary adenoma patients , most of which were young people with an average age of 33.415.3 years . patients had no sex bias or no statistical difference of general clinical information such as age or sex among all 3 types , consistent with results of a previous study . due to these critical physiological functions , pituitary adenoma leads to various neural and endocrine symptoms , including optic nerve impairment , hypothyroidism , underdevelopment of sexual characters , growth hormone secretion insufficiency , adrenal cortex dysfunction , and hyperprolactinemia [ 1719 ] . the invasive nature of pituitary adenoma makes it impossible to completely remove tumor tissues by surgical resection , causing the high recurrence rate of pituitary adenoma . studies have revealed a complicated mechanism underlying pathogenesis of invasive pituitary adenoma , which is induced by multiple factors involved in the neural - endocrine - immune regulatory network . currently , there are few effective and easy methods for clinical diagnosis of pituitary adenoma and prediction of tumor prognosis . cirp was the first - discovered cold - stress or cold - shock protein in mammalian cells . recent studies found highly - conserved structures of cirp in terms of its nucleotide or amino acid sequence , accompanied by high homogeneity . although first discovered under cold - induced cell growth inhibition , cirp was later found to participate in multiple stress responses , including those induced by ultraviolet radiation , osmotic alternation , or hypoxia / ischemia , in addition to critical roles in embryonic development , reproduction , and tumors ; therefore , having valuable prospective clinical applications . previous studies have reported cirp as a novel oncogene that can facilitate the proliferation of tumor cells via its up - regulation of expression in various tumor cells and further inhibition of oxidative - induced tissue damage , thereby exerting a trophic role in oncogenesis . thus , this study focused on cirp to determine if it can work as an index for pathogenesis and progression of pituitary adenoma . results showed that cirp protein was expressed in all 3 types of pituitary adenoma , but with different expression levels . non - invasive pituitary adenoma had the lowest expression of cirp protein , which was significantly elevated when adenoma was invading peripheral neural tissues . it is important that both mrna and protein of cirp have the highest levels in recurrent pituitary adenoma , suggesting a close relationship between invasion and proliferation of pituitary adenoma and cirp expression , whose significant elevation clearly indicates post - op recurrent of pituitary adenoma . the mechanism of cirp in tumor proliferation and invasion has not yet been reported , making it necessary to further elucidate the role of cirp in pathogenesis of pituitary adenoma . due to the relatively small sample size involved in this study , further research should be conducted in an expanded sample to further validate the expressional profile of cirp in pituitary adenoma tissues . cirp can work as an index for evaluating invasion and proliferation of pituitary adenoma , and may indicate the possibility of post - op tumor recurrence . the inhibition of cirp expression can be a treatment strategy against recurrent pituitary adenoma , although the detailed functional mechanism of cirp in pathogenesis of pituitary adenoma remains to be fully defined .
backgroundthe aim of this study was to detect the expression of cold - inducible rna - binding protein in pituitary adenoma and to determine its effects on tumor recurrence.material/methodswe collected a total of 60 post - op samples collected from pituitary adenoma patients ( including 20 cases of invasive pituitary adenoma , 20 cases of non - invasive adenoma , and 20 cases of non - invasive recurrent adenoma ) admitted in our hospital . 
 both protein and mrna levels of cirp in 3 types of pituitary adenoma samples were quantified by western blotting and real - time pcr , respectively.resultswestern blotting revealed significantly elevated cirp expression levels in invasive pituitary adenoma compared to non - invasive tumors , with statistical significance ( p<0.05 ) . 
 recurrent pituitary adenoma expressed significantly higher cirp levels compared to non - recurrent tumors ( p<0.05 ) . 
 real - time pcr for cirp mrna obtained consistent results : transcript levels were significantly higher in invasive pituitary adenoma compared to non - invasive adenoma ( p<0.05 ) ; recurrent adenoma also had significantly higher cirp mrna levels compared to non - recurrent tumors ( p<0.05 ) . among all 3 types of pituitary adenoma 
 , recurrent tumors had the highest levels of cirp mrna and protein.conclusionsthe expression of cirp in pituitary adenoma is closely related with tumor proliferation and invasion , and its significantly elevated expression level indicates post - op recurrence .