Oncogenic reg IV is a novel prognostic marker for glioma patient survival
- Qi Wang†1,
- Jianping Deng†2,
- Jun Yuan†3,
- Liang Wang2,
- Zhenwei Zhao2,
- Shiming He2,
- Yongsheng Zhang3Email author and
- Yanyang Tu1Email author
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 19 June 2012
Published: 19 June 2012
The aberrant expression of regenerating islet-derived family member, 4 (Reg IV) has been found in various human cancers. However, the roles of Reg IV gene and its encoding product in human glioma have not been clearly understood. Therefore, the aim of this study was to investigate the clinicopathological significance of Reg IV expression in glioma.
Reg IV mRNA and protein expression in human gliomas and non-neoplastic brain tissues were respectively detected by real-time quantitative RT-PCR assay, Western blot, and immunohistochemistry. The association of Reg IV immunostaining with clinicopathological factors and prognosis of glioma patients was also statistically analyzed.
Reg IV mRNA and protein expression levels in glioma tissues were both significantly higher than those in the corresponding non-neoplastic brain tissues (both P < 0.001). Additionally, the increased Reg IV immunostaining in glioma tissues was significantly associated with advanced pathological grade (P = 0.008). Reg IV protein up-regulation was also significantly correlated with low Karnofsky performance score (KPS) (P = 0.02). Moreover, the overall survival of patients with high Reg IV protein expression was dramatically shorter than those with low Reg IV protein expression (P < 0.001). Multivariate Cox regression analysis further confirmed that Reg IV expression was an independent prognostic factor for patients with gliomas (P = 0.008).
These convinced evidences suggest for the first time that Reg IV might accelerate disease progression and act as a candidate prognostic marker for gliomas.
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Human glioma represents the most common malignancy in central nervous system for both children and adults. According to the World Health Organization (WHO) classification, gliomas are divided into well-differentiated low grade astrocytomas [WHO grade I ~ II], anaplastic astrocytomas (WHO grade III) and glioblastoma multiforme (GBM, WHO grade IV) , which especially represents the most aggressive and the most lethal type of brain tumor. In recent years, there have been great progress occurred in therapeutic technologies, such as surgery, radiotherapy, photodynamic therapy [2, 3], and chemotherapy; however, the clinical outcome of glioma patients remains poor, with an average patient life expectancy of only 15 months after diagnosis for GBM patients . The reason for this unfavorable prognosis is that tumor cells in GBM tissues are not only highly proliferative but also readily invade surrounding brain structures, thereby making complete surgical resection practically impossible. Hence, molecular characteristics responsible for the tumor invasion process and the suboptimal response to conventional therapies are potential prognostic and therapeutic markers for human gliomas.
Human regenerating (Reg) genes, initially identified from regenerating rat pancreatic islets, encode five small-secreted proteins (Reg Iα, Reg Iβ, Reg IIIα, Reg IIIβ and Reg IV) with similar structure to the proteins of the calcium-dependent lectin (C-type lectin) superfamily . Reg proteins act as inflammation-modifying acute phase reactants and mucosal repair stimulating factors in the gastrointestinal tract, growth factors and anti-apoptotic survival signal mediators for pancreatic islet cells and neurons . As the most recently discovered member of the human Reg family proteins, Reg IV has been demonstrated to enhance cell proliferation and inhibit apoptosis by inducing phosphorylation of epidermal growth factor receptor (EGFR) (Tyr992, Tyr1068) and Akt (Thr308, Ser473) . In normal human body, the expression of Reg IV mRNA is restricted to gastrointestinal tract, pancreas, prostate, and testes . Normal parietal cells in stomach stain for Reg IV, whereas goblet cells of the ventricular mucosa in areas displaying intestinal metaplasia and in Barrett’s esophagus showed robust accumulation of Reg IV . In normal intestinal mucosa, goblet cells show a weak Reg IV staining, while the mucosa in inflammatory bowel disease is strong Reg IV positive . The aberrant expression of Reg IV has been found in various human cancers, including gastric cancer, colorectal cancer, ductal cancer of the pancreas, gallbladder carcinoma, prostate cancer, and adenoid cystic carcinoma in the salivary gland [10–15]. Recent studies also suggested that Reg IV may play an important role in initiating tumorigenesis, and its detection might be useful in the early diagnosis of tumor formation. However, the role of Reg IV gene and its encoding product in human glioma has not been clearly understood. To address this problem, Reg IV expression at mRNA and protein levels in human glioma and non-neoplastic brain tissues were respectively measured by real-time quantitative RT-PCR assay, Western blot, and immunohistochemistry. The association of Reg IV immunostaining with clinicopathological factors or prognosis of glioma patients was also statistically analyzed.
Materials and methods
Patients and tissue samples
This study was approved by the Research Ethics Committee of Tangdu Hospital, Fourth Military Medical University, P. R. China. Written informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards.
Clinicopathological features of 128 patients with gliomas
Mean age (year)
Gross total resection
Radiotherapy and Chemotherapy combination
Real-time quantitative RT-PCR
The mRNA expression of Reg IV in glioma and non-neoplastic brain tissues was detected by real-time quantitative RT-PCR analysis according to the conventional protocols of Tangdu hospital . Especially, the primers were designed as follows: for human Reg IV, forward primer, 5'- TGC ACG ACC CAC AGA AGA G -3', and reverse primer, 5'- GAC TTG CCA GAC CAG GAT CT -3'; for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward primer, 5'- CCC ACT CCT CCA CCT TTG AC-3', and reverse primer, 5'-ATG AGG TCC ACC ACC CTG TT-3'. Each sample was examined in triplicate and the amounts of the PCR products produced were non-neoplasticized to GAPDH which served as internal control.
Western blot analysis
The protein expression of Reg IV in glioma and non-neoplastic brain tissues was detected by Western blot analysis according to the conventional protocols of Tangdu hospital [16–18]. Briefly, the tissues were harvested by scraping and lysed using modified radioimmunoprecipitation assay buffer (50 mM Tris–HCl, pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitor cocktail complete. The protein content was determined according to Bradford’s method, with bovine serum albumin used as a standard. Equal amounts of protein were separated electrophoretically on 7.5% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Roche; Basel, Switzerland). The membranes were probed with a goat anti-human Reg IV polyclonal antibody (1:25, R&D Systems, Abingdon, UK). The expression level of Reg IV was determined by incubating the membranes with horseradish peroxidase-conjugated anti-goat immunoglobulin G (1:1000 dilution) and enhanced chemiluminescence reagent (Pierce; Minneapolis, MN, USA), according to the manufacturers’ suggested protocols. The membranes were stripped and reprobed with an anti-β-actin mouse monoclonal antibody (1:1000 dilution: Sigma; St Louis, MO, USA) as a loading control. Densitometry was performed using ImageJ software (http://rsb.info.nih.gov/ij/) from National Institutes of Health (NIH; Bethesda, MD, USA). We evaluated the expression of Reg IV as an optical densitometry (OD) ratio that was scored as the densitometry of Reg IV relative to the densitometry of β-actin.
The immunostaining of Reg IV protein in glioma and non-neoplastic brain tissues was detected by Immunohistochemistry assay according to the conventional protocols of Tangdu hospital [16–18]. Briefly, specimens were blocked with phosphate-buffered saline (PBS) containing 5% normal horse serum (Vector Laboratories Inc., Burlingame, CA, USA) following peroxidase blocking with 0.3% H2O2/methanol for 30 min. All incubations with goat anti-human Reg IV polyclonal antibody (1:25, R&D Systems, Abingdon, UK) were carried out overnight at 4°C. Then the specimens were briefly washed in PBS and incubated at room temperature with the anti-goat antibody and avidin-biotin peroxidase (Vector Laboratories Inc., Burlingame, CA, USA). The specimens were then washed in PBS and color-developed by diaminobenzidine solution (Dako Corporation, Carpinteria, CA, USA). After washing with water, specimens were counterstained with Meyer’s hematoxylin (Sigma Chemical Co., St Louis, MO, USA). Non-neoplastic brain tissues were used as control tissues and non-immune IgG was also used as negative control antibody for immunohistochemical staining. The immunohistochemical reaction was performed simultaneously for all slides.
Assessment of immunohistochemical staining was evaluated by two independent pathologists. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of Reg IV positive-staining cells showing immunoreactivity in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1-10%), 2 (11-50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). A final immunoreactivity scores (IRS) was obtained for each case by multiplying the percentage and the intensity score. The median of IRS for all examined samples was 5.3. Thus, the Reg IV protein expression levels were further analyzed by classifying IRS values as low (based on a IRS value less than 5.3) and as high (based on a IRS value greater than 5.3).
All computations were carried out using the software of SPSS version13.0 for Windows (SPSS Inc, IL, USA). Data were expressed as means ± standard deviation (SD). The analysis of variance (ANOVA) was used to determine the statistical differences among the groups. Spearman rank correlation is used to analyze the correlation between Reg IV mRNA expression and Reg IV protein expression. A life table was calculated according to the Kaplan-Meier method. Hazard ratios for the time-to-event endpoint were estimated using the multivariate Cox regression analysis in a forward stepwise method to evaluate the effect of multiple independent prognostic factors on survival outcome. Differences were considered statistically significant when p was less than 0.05.
Reg IV mRNA and protein expression in human glioma tissues
Reg IV immunostaining and its association with clinicopatholigcal features of gliomas
Association of Reg IV expression in human glioma tissues with different clinicopathological features
No. of cases
Reg IV expression
High (n, %)
Low (n, %)
Prognostic value of reg IV expression in overall survival of patients with gliomas
Cox multivariate analysis
95% confidence interval
Extent of resection
Type of adjuvant treatment
Reg IV expression
Understanding of the molecular alterations that occur during tumorigenesis, and identification of novel markers for cancer diagnosis and novel targets for treatment, may be important for the improvements in tumor diagnosis, treatment and prevention. This study was focus on human glioma, which is an aggressive tumor with heterogeneous tumor biology, high invasiveness, rapid tumor cell proliferation and poor prognosis. Several genetic and molecular mechanisms of tumorigenesis in gliomas have been explained; however, factors affecting this tumor progression remain unclear. As results of our analysis, there are four points of findings. Firstly, Reg IV was up-regulated in human glioma tissues compared with non-neoplastic brain tissues at both mRNA and protein levels; Secondly, the increased Reg IV expression in glioma tissues was significantly correlated with advanced tumor progression and aggressive clinicopathological features; Thirdly, the results of Kaplan-Meier analyses shown that glioma tissues with high Reg IV expression tend to have poorer overall survival. Finally, the multivariate analysis clearly demonstrated that the high expression of Reg IV was a statistically significant risk factor affecting overall survival in glioma patients, suggesting that Reg IV expression could be a valuable marker of glioma progression and prognosis. To our knowledge, this is the first study to analyze the expression patterns and clinical significance of Reg IV at transcriptional and translational levels in a large number of glioma patients.
Reg genes encode five multifunctional small-secreted proteins, which can act as acute phase reactants, lectins, or antiapoptotic or growth agents . As a novel member of the Reg family, human Reg IV is mapped to chromosome 1q12-q21, whose cDNA contains an open reading frame of 477 bp encoding a peptide of 158 amino acids with a predicted molecular mass of 18 kD . Reg IV functions as a potent activator of the EGFR/Akt/AP-1 signaling pathway in several types of cancer cells and increases the expression of Bcl-2, Bcl-xl, and surviving proteins, related to the inhibition of apoptosis, suggesting that it may act as a tissue mitogen or play a role in the cell growth . Additionally, Reg IV has been found to be colocalized with Ki-67, indicating that it may be involved in the proliferative process of epithelial cells . Besides this, it also contributes to liver metastasis by inducing the expression of matrix metalloproteinase 7 . In recent year, accumulating studies on Reg IV had reported its presence and overexpression in numerous cancers. The up-regulation of Reg IV in colorectal adenomas was testified by several different research groups [10, 24, 25]; Zhang et al. also indicated that Reg IV might play an important role in initiating colorectal adenoma, and its detection might be useful in the early diagnosis of colorectal adenoma formation. In gastric carcinogenesis, Zheng et al. detected the increased expression of Reg IV from gastric intestinal metaplasia to adenoma, but the decreased expression during the malignant transformation of gastric epithelial cells. These findings revealed that Reg IV expression should be considered as a good biomarker for gastric precancerous lesions. In prostate cancer, Hayashi et al. found the higher serum Reg IV concentration compared with control individuals, indicating that serum Reg IV represents a novel biomarker for this disease . In line with these previous studies, our data in the present study demonstrated the up-regulation of Reg IV mRNA and protein in human glioma tissues. Interestingly, we also determined that the Reg IV mRNA expression was closely correlated with its protein expression, suggesting that the up-regulation of Reg IV in gliomas may be primarily caused by transcriptional activation. Additionally, its overexpression was associated with advanced pathological grade and low KPS. These results confirmed the contribution of Reg IV expression to the aggressive progression of human gliomas.
Given above intriguing observation correlating Reg IV up-regulation with advanced tumor progression in human gliomas, we were further interested in studying a potential prognostic value of Reg IV for this malignancy. As the results, overall survival after surgical resection among glioma patients in Reg IV-high group was poorer than those in Reg IV-low group. However, there are some different findings on the relationship between Reg IV expression and survival of patients with cancer. For example, Tamura et al. revealed that the positive Reg IV expression might be independently associated with a favorable prognosis in patients with advanced gallbladder carcinoma. In gastric cancer, no statistically significant prognostic effect of Reg IV was found . These different reports suggest that the contribution of Reg IV expression to the clinical outcome of patients may be different according to the type of malignancy.
In conclusion, our data offer the convince evidence for the first time that Reg IV might accelerate disease progression and act as a candidate prognostic marker for human gliomas. Further elucidation on the roles of Reg IV as tumor promoter in gliomas is worth to be done.
This work was funded by National Natural Science Foundation of China (NO. 81101736).
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