Currently, the role of p53-induced RING-H2 protein (PIRH2) in the development of hepatocellular carcinoma (HCC) remains unknown. The objective of this retrospective study was to investigate the expression of PIRH2 and its relation to prognosis in patients with HCC after hepatic resection.
Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and Western blot analyses were used to detect expression levels of PIRH2 in 30 samples of HCC tissue and paracarcinomatous liver tissue (PCLT) and in 5 samples of normal liver tissue (NL). In addition, immunohistochemical analysis was performed on 122 HCC specimens and follow-up information data from those patients were reviewed.
Both messenger RNA and protein expression levels of PIRH2 were elevated significantly in HCC tissues compared with PCLT and NL tissues. The increased PIRH2 expression was correlated with vein invasion, Edmondson-Steiner grade, TNM stage, and multiple tumor nodes (P<.05). It is noteworthy that the patients with HCC who had high PIRH2 expression had shorter overall survival and disease-free survival than the patients who had low PIRH2 expression (median survival, 280 days vs 372 days; P = .0002; median disease-free survival, 220 days vs 310 days; P = .0016). Multivariate Cox regression analysis revealed that high PIRH2 expression was an independent prognostic factor for patients with HCC (relative risk, 1.792; P = .009).
Hepatocellular carcinoma (HCC) is 1 of the most common human cancer types in the world, especially in China. There are great numbers of new cancer cases annually (626,000), and HCC ranks sixth among the most common malignancies worldwide and results in almost the same numbers of deaths (598,000).1-3 With the improvement in liver surgery techniques, hepatic resection for HCC increasingly has become safer, and the operative mortality rate has decreased to <5% in large clinical centers.4-6 However, the long-term survival of patients with HCC remains unsatisfactory because of the high incidence of recurrence and metastasis after hepatic resection, with a 5-year survival rate of 20% to 30% reported in the literature.7, 8 To predict recurrence, metastasis, and prognosis in patients with HCC after hepatic resection is a significant clinical issue. Although the identification of many prognostic markers of HCC, such as vascular endothelial growth factor, osteopontin, and transforming growth factor-β, resulted from extensive clinical as well as basic research efforts, the available markers still are not able to accurately predict the survival of patients with HCC.9-11 Thus, exploring new prognosis markers of HCC is of great importance.
The ubiqutin-proteasome pathway is important in mediating the degradation of many cellular proteins. It plays a critical role in the regulation of cell cycle control, DNA repair, and apoptosis.12, 13 Recent studies have demonstrated that E3 ubiquitin ligases frequently are overexpressed in many malignant tumors and are involved in cancer development. Increased expression of E3 ligases also reportedly is correlated with chemoresistance and a poor prognosis in patients with cancer.14, 15 Thus, E3 ubiquitin ligases have been considered as potential cancer drug targets and prognostic biomarkers.16-19 p53-Induced RING-H2 protein (PIRH2) is a newly identified E3 ubiquitin ligase that promotes p53 degradation.20, 21
The PIRH2 gene encodes a RING-H2 domain-containing protein that has intrinsic ubiquitin-protein ligase activity. It has been demonstrated that PIRH2 can interact with and promote p53 ubiquitination independent of mouse double-minute 2 (MDM2).22, 23 It is noteworthy that, in recent studies, the PIRH2 gene was overexpressed in lung cancer and prostate cancer, and overexpression of PIRH2 plays a potential role in the development of cancer.24, 25 However, the expression profile and function of PIRH2 in HCC currently remains unknown. In the current study, we sought to determine the expression level of PIRH2 in HCC and analyzed the correlation between PIRH2 expression and clinicopathologic characteristics. Furthermore, the relation between PIRH2 expression and survival in patients with HCC after hepatic resection was analyzed to explore the prognostic value of this E3 ligase.
MATERIALS AND METHODS
Patients and Samples
Specimens from 30 patients with HCC and 5 normal liver samples from patients with liver cavernous hemangioma (controls) were immediately frozen in liquid nitrogen and stored at −80°C after hepatectomy. In addition, specimens from 122 patients with HCC were collected for immunohistochemistry analysis, which included 105 men and 17 women with a median age of 48 years (range, 24-71 years). All specimens were obtained from patients who underwent hepatectomy at the Department of General Surgery, Xiangya Hospital of Central South University between October 2005 and September 2007. Specimens were paraffin embedded and stained with hematoxylin and eosin. Clinicopathologic parameters, included sex, age, greatest tumor dimension, the number of tumor nodes, tumor capsule, Edmondson-Steiner grade, vein invasion, and TNM classification (according to the International Union Against Cancer, 2003).26 The diagnoses were confirmed by histopathologic study. Written informed consent was obtained from these patients, and the protocol for this study was approved by the Ethics Committee of the Central South University.
Prior informed consent was obtained from patients for the collection of liver specimens in accordance with the guidelines of Xiangya Hospital. The study protocols were approved by the Ethics Committee of the Central South University, People's Republic of China.
Total RNA was extracted from frozen tissue specimens (50-100 mg) using Trizol reagent (Invitrogen, Carlsbad, Calif), according to the instructions provided by the manufacturer. The quality of RNA was determined by using the agarose gel analysis, and the concentration was measured with a spectrophotometer (Biochrom Ltd, Cambridge, England). The expected size of PIRH2 is a 430-base-pair fragment. The primers, which were synthesized by Boshang Biotechnology Company (Shanghai, People's Republic of China), were as follows: PIRH2-forward, 5′-GCGAGCACTATGACAGAGGATG-3′; and PIRH2-reverse, 5′-GGC AAGACATGAGCAACAACAC-3′. Expression of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was determined as a control. Total RNA (1 μg) was reverse transcribed by Avian Myeloblastosis virus reverse transcriptase (RT) and Oligo dT-Adaptor Primer (Promega, Madison, Wis) according to the manufacturer's protocols and under the following conditions: 30°C for 5 minutes, 40°C for 15 minutes, 99°C for 5 minutes. Re product (2 μL) was amplified on a DNA Thermal cycler (Perkin-Elmer, Shelton, Conn) by using TaKaRa Ex Taq HS DNA polymerase (Takara, Dalian, People's Republic of China) under the following conditions: 94°C for 5 minutes; then 28 cycles at 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute; and an extension at 72°C for 7 minutes. Then, the polymerase chain reaction (PCR) products (5 μL) were electrophoresed on a 2% agarose gel, and the intensity of bands was quantified by using the Eagle Eye II laser densitometry program (Strategene, La Jolla, Calif). The level of PIRH2 messenger RNA (mRNA) expression was expressed as the intensity of the PCR product bands from target sequences relative to the intensity from the GAPDH gene. PCR experiments were done in triplicate.
PIRH2 and GAPDH were used as target gene and internal loading control, respectively. The primers, which were synthesized by Boshang Biotechnology Company (Shanghai, People's Republic of China), were as follows: PIRH2-forward, 5′-GACAGCTGGATGATGAAGTAGCAC A-3′; PIRH2-reverse, 5′-CTCGTCATTGCTGATCCAGTGAA-3′; GAPDH-forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; and GAPDH-reverse, 5′-TGGTGAAGACGCCA GTGGA-3′. Quantitative real-time RT-PCR (QRT-PCR) amplification was conducted in a 25-μL reaction using the SYBR Premix Ex TaqTM (Takara, Dalian, People's Republic of China). Temperature conditions consisted of 1 step for 10 seconds at 95°C followed by 40 cycles at 95°C for 5 seconds and at 64°C for 31 seconds. All amplification reactions were performed in triplicate. PCR product quality was monitored using post-PCR melt-curve analysis. Reactions were carried out in a 96-well plate using the ABI Prism 7300 Real-Time PCR System (Applied Biosystems, Foster City, Calif).
Western Blot Analysis
Total protein was extracted from fresh tissue specimens, and the concentration of protein was determined by using a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill). Total protein (100 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Sigma Chemical Company, St. Louis, Mo). The blotted membranes were incubated with goat antihuman PIRH2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) diluted at 1: 500 and rabbit antihuman p53 polyclonal antibody (Santa Cruz Biotechnology) diluted at 1: 500, respectively. After washing, the membranes were incubated with a 1:3000 dilution of horseradish peroxidase-linked rabbit antigoat antibody (Santa Cruz Biotechnology) and horseradish peroxidase-linked goat antirabbit antibody (KPL, Gaithersburg, Md), respectively. The blots were developed by enhanced SuperSignal West Pico chemiluminescence (Pierce). Actin levels also were determined by using the specific antibody (Santa Cruz Biotechnology) as a loading control. All experiments were carried out in triplicate, and the levels of PIRH2 protein were quantified by using a densitometer (Beckman, South Pasadena, Canada).
Tissue sections (4 μm thick) were cut for immunohistochemical staining. The tissue sections were baked at 60°C for 36 hours, deparaffinized in xylene, and rehydrated through a graded series of ethanol. Subsequently, 3% hydrogen peroxide was used to block the endogenous peroxidases for 30 minutes, and the sections were subjected to microwave heat-induced antigen retrieval in 1 mmol/L ethylene diamine tetraacetic acid, pH 8.0, at high power twice for 10 minutes each. The sections were incubated first with normal rabbit serum to reduce nonspecific binding and then with specific antibodies (goat antihuman PIRH2 polyclonal antibody; Santa Cruz Biotechnology) at 4°C for 12 hours. The second antibody was incubated for 30 minutes at 37°C. The streptavidin-peroxidase complex system (Zhongshan Goldenbridge Biotechnology, Beijing, People's Republic of China) was used according to the manufacturer's instruction for 30 minutes at 37°C. The tissues were observed by applying 3,3-diaminobenzidine tetrahydrochloride (Zhongshan Goldenbridge Biotechnology, Beijing, People's Republic of China) for 3 minutes. Tissues were counterstained with hematoxylin and mounted for examination. Control slides were probed with normal goat serum under the same experimental conditions. The intensity of cytoplasmic staining was scored from 0 to 3+ by comparing it with the intensity in positive controls, as described previously.24, 27 The intensity of PIRH2 protein staining in HCC specimens was classified using the following 4-point scale: 0, ≤10% positive cells; 1+, 11% to 25% positive cells; 2+, 26% to 50% positive cells; and 3+, ≥51% positive cells. The expression levels of PIRH2 protein, thus, were divided into low expression (0 or 1+) and high expression (2+or 3+). Immunohistochemical analysis and scoring were performed by 2 independent investigators.
Follow-up data were obtained by reviewing the hospital records, direct communication with the patients after hepatic resection for all 122 patients. The follow-up period was defined from the date of surgical excision of the tumor to the date of death or last follow-up. Deaths from other causes were treated as censored cases. Patients whose deaths clearly were documented as attributable to HCC were considered as deaths from that disease; other deaths were not considered to be caused by HCC. The disease-free survival was defined as the length of time after hepatectomy for HCC during which a patient survives with no sign of HCC. The follow-up time ranged from 30 to 870 days, with a median follow-up time of 320 days. To determine factors influencing survival after operation, 10 conventional variables together with PIRH2 expression were tested in all 122 patients: age (≤60 years vs >60 years), sex, cirrhosis (presence vs absence), serum AFP level (≤20 ng/mL vs >20 ng/mL), Edmondson-Steiner grade (I-II vs III-IV), capsule (presence vs absence), size of the tumor (≤5 cm vs >5 cm), the number of tumor nodes (solitary vs multiple), vein invasion (absence vs presence), TNM stage (I-II vs III) and PIRH2 protein expression level (high vs low).27, 28
SPSS13.0 for Windows (Chicago, Ill, USA) was used for statistical analysis. The independent sample t test was used to compare the PIRH2 mRNA and protein expression levels between tumor tissue and paracarcinomatous liver tissue (PCLT) or normal liver tissue (NL). A correlation between PIRH2 expression levels and clinicopathologic characteristics in patients with HCC was examined. Spearman correlation coefficients were used to analyze the correlations between expression levels of PIRH2 mRNA and protein. The chi-square test was used to analyze the expression of PIRH2 protein levels in 122 patients with HCC between tumor tissues and PCLT, and Spearman rank-correlation analysis was used to analyze the correlation between PIRH2 expression levels and clinicopathologic characteristics in patients with HCC. Survival curves were plotted using the Kaplan-Meier method and were analyzed using the log-rank test. The Cox proportional hazards regression model was used to identify factors that were associated independently with survival. All tests were 2-tailed, and all P values <.05 was considered statistically significant.
Increased Expression Levels Of p53-Induced RING-H2 Protein Messenger RNA and Protein in Hepatocellular Carcinoma
The expression of PIRH2 mRNA was detected in all of 30 fresh HCC tissues, PCLTs, and NL tissues by RT-PCR. HCC tissues expressed significantly higher mRNA levels of PIRH2 than PCLTs (0.62 ± 0.23 vs 0.46 ± 0.29; P = .020) and NL tissues (0.62 ± 0.23 vs 0.38 ± 0.11; P = .029) (Fig. 1). Further QRT-PCR performed in the same samples confirmed that PIRH2 mRNA in HCC was significantly higher than that in PCLTs (P < .001) and NL tissues (P < .001) (Fig. 1). PIRH2 proteins also were detected in all samples by Western blot analysis. Consistent with mRNA expression, protein expression of PIRH2 in HCC tissues also was significantly higher than that in the corresponding PCLTs (0.88 ± 0.30 vs 0.66 ± 0.26; P = .004) and NL tissues (0.88 ± 0.30 vs 0.45 ± 0.21; P = .005) (Fig. 1). In addition, the corresponding p53 protein expression in the same samples of HCC tissues was significantly lower than that in matched PCLTs (0.35 ± 0.07 vs 0.59 ± 0.12; P < .001) and NL tissues (0.35 ± 0.07 vs 0.61 ± 0.09; P < .001) (Fig. 1). Furthermore, there was a significantly positive correlation between the expression level of PIRH2 mRNA and protein in HCC samples by Spearman correlation coefficient analysis (r = 0.930; P < .001) (Fig. 1).
Immunohistochemical staining revealed a cytoplasmic distribution of PIRH2. The PIRH2 protein was detected in 109 of 122 HCC tissues and in 33 of 122 PCLTs (Fig. 2). The positive expression rate of PIRH2 was significantly higher in HCC tissues than that in PCLTs (89.34% vs 27.05%; P < .001).
Correlations Between p53-Induced RING-H2 Expression Levels and Clinicopathologic Parameters in Hepatocellular Carcinoma
In Tables 1 and 2, the immunohistochemical staining results indicated that the up-regulation of PIRH2 protein expression was correlated strongly with Edmondson-Steiner grade, vein invasion, multiple tumor nodes, and TNM classification. The PIRH2 protein expression levels in tumors at TNM stage III were significantly higher than those in tumors at TNM stage I (P = .004) or TNM stage II (P = .047). Although the expression levels of PIRH2 in tumor tissues from patients with TNM stage I disease were lower than the levels in tissues from patients with TNM stage II disease, the difference was not statistically different (P = .108). There was no significant association between PIRH2 expression and the other clinicopathologic parameters.
Table 1. Correlations Between p53-Induced RING-H2 Protein Expression Levels and Clinicopathologic Characteristics of 122 Patients With Hepatocellular Carcinoma*
No. of Patients
PIRH2 indicates p53-induced RING-H2 protein.
The correlations between PIRH2 protein expression levels and clinicopathologic characteristics were evaluated by using Spearman rank-correlation analysis.
Tumor size, cm
No. of tumor nodes
Table 2. Correlations Between p53-Induced RING-H2 Protein Expression Levels and Tumor-Lymph Node-Metastasis Classification in 122 Patients With Hepatocellular Carcinoma*
Relation Between the p53-Induced RING-H2 Expression Level and Prognosis
To examine the correlation between PIRH2 expression levels and prognosis, patients with HCC were divided into the low PIRH2 expression group (immunohistochemistry score, 0 and 1+; n = 44) and the high PIRH2 expression group (immunohistochemistry score, 2+ and 3+; n = 78). The PIRH2 expression level and the prognosis of patients with HCC were analyzed by using the Kaplan-Meier method. The results indicated that patients who had high PIRH2 expression had a shorter overall survival and disease-free survival than patients who had low PIRH2 expression (median survival, 280 days vs 372 days; P = .0004; median disease-free survival, 220 days vs 310 days; P = .0016) (Fig. 3). The multivariate Cox regression analysis indicated that high PIRH2 expression (relative risk [RR], 1.792; P = .009) and multiple tumor nodes (RR, 1.554; P = .031) were independent prognostic factors for survival. The other 9 clinicopathologic parameters did not add any independent prognostic information (Table 3).
Table 3. Cox Regression Analyses of Overall Survival, p53-Induced RING-H2 Protein Expression Levels, and Clinicopathologic Parameters
Recently, it was reported that PIRH2 interacts with p53 and promotes the ubiquitination of p53 independent of Mdm2. Expression of PIRH2 decreases the level of p53, and inhibition of PIRH2 expression increases the level of p53. PIRH2 functions to suppress p53 activity, including p53-dependent transactivation and growth inhibition.20 Given the importance of p53 as a tumor suppressor,29-37 degradation of p53 would implicate a role for PIRH2 in carcinogenesis and in the development of cancer. Previous studies revealed that the PIRH2 gene is overexpressed in lung cancer and prostate cancer24, 25; however, the correlation between its expression and patient prognosis has not been documented, and the expression profile and function of PIRH2 in HCC currently remain unknown. We hypothesized that overexpression of PIRH2 plays a potential role in the development of cancer, and it may be a novel prognostic marker for HCC. Thus, we performed the current study to investigate the correlation between PIRH2 express ion and prognosis in patients with HCC after hepatic resection. In this study. first, we examined PIRH2 expression profiles in HCC and the correlations between its expression and clinicopathologic parameters and prognosis. Our data revealed that both mRNA and protein levels of PIRH2 were significantly higher in HCC tissues than in the corresponding PCLTs and NL tissues. Moreover, we further found that the corresponding p53 protein expression in the same samples of HCC tissues was significantly lower than that in matched PCLT and NL tissues, confirming that expression of PIRH2 decreases the level of p53 in HCC.
The prediction of recurrence, metastasis, and prognosis in patients with HCC after hepatic resection is an important clinical issue that could determine the surgical therapeutic regimen. We observed that increased expression of PIRH2 in HCC was correlated positively with tumor vein invasion, multiple tumor nodes, poor cell differentiation, and TNM classification. It is well known that vein invasion, multiple tumor nodes, poor cell differentiation, and TNM classification are highly correlated with invasion and metastasis and with a poor prognosis in patients who have HCC.38-41 Our current study indicates that the up-regulated expression of PIRH2 is correlated with poor survival for patients with HCC. It is noteworthy that, whereas our univariate analysis indicated that high PIRH2 expression, multiple tumor nodes, and TNM classification were risk factors for prognosis in patients with HCC, our multivariate Cox regression analysis indicated that high PIRH2 expression and multiple tumor nodes were the only independent risk factors of prognosis for patients with HCC. Thus, our data indicate that PIRH2 expression can serve as a prognostic marker for patients with HCC. In summary, this study demonstrated that increased PIRH2 expression levels were correlated with a poor prognosis in patients with HCC, indicating that PIRH2 may serve as a novel prognostic marker for HCC after hepatic resection.
We thank Feng Fang for collecting survival and histopathologic data on patients with hepatocellular carcinoma. We also thank Wei Wang for article preparation.
Conflict of Interest Disclosures
Supported by a grant from the National Key Technology R&D Program of China (2001BA703B04 and 2004BA703B02), a grant from the National Basic Research Program of China (2004CB720303), a grant from the National Natural Science Foundation for Distinguished Young Scholars of China (30328028), and a grant from the National High Technology Research and Development Program of China (2006AA02Z4B2).