Prohibitin-1 (PHB), also known as PHB1, is a 32 kDa evolutionarily conserved protein that shares a domain with stomatin/prohibitin/flotillin/HflK/C (Merkwirth and Langer 2009). PHB has been reported to localize to the mitochondria, nucleus and plasma membrane of certain mammalian cell lines (Fusaro et al., 2003). In addition, PHB has been found in plasma, although the source of circulating PHB is unclear. PHB plays an important role in cell cycle control, differentiation, apoptosis and senescence and determines whether the cell should either proliferate or initiate the process of programmed cell death (Mishra et al., 2006). Furthermore, PHB is an essential element in embryonic development because the total ablation of PHB in mice results in early embryonic lethality (He et al., 2008). Recent functional studies have defined a critical role for mitochondria-localized PHB in cellular homeostasis (Merkwirth and Langer 2009); moreover, the interactions of PHB with E2F, pRb and p53 can also govern its action (Fusaro et al., 2003). The role of PHB in cancer cell proliferation and/or tumour suppression remains controversial. Many reports have shown evidence that PHB has anti-tumourigenic activity in prostate (Dart et al., 2009), gastric (Liu et al., 2009) and liver (Ko et al., 2010) cancer. PHB overexpression results in the inhibition of prostate cancer cell growth and the knockdown of PHB by siRNA accelerates tumour growth (Dart et al., 2009). However, studies also suggest that the PHB plays a protumorigenic role. PHB was shown to be necessary for the activation of C-Raf by the oncogene Ras in HeLa cells (Rajalingam et al., 2005). Thus, the relationship between PHB and cancer is only beginning to be elucidated and requires further investigation.
Nasopharyngeal carcinoma (NPC) is endemic to Southern China, Southeast Asia and North Africa. Using suppression subtractive hybridization and a cDNA microarray, we previously identified LPLUNC1 (long palate, lung, nasal epithelium clone 1) (C20orf114) as a tissue-specific gene in nasopharyngeal epithelia, which is downregulated in NPC (Zhang et al., 2003). LPLUNC1 is a secreted protein (Bingle et al., 2010) that belongs to the bactericidal permeability-increasing protein/lipid binding protein family and that can bind to bacterial lipopolysaccharides. Importantly, our studies have shown that LPLUNC1 can inhibit the proliferation of NPC cells (Yang et al., 2007). In this study, we used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and matrix-assisted laser desorption/ionization time of flight (MALDI–TOF/TOF) mass spectrometry to detect proteins that are differentially regulated following the overexpression of LPLUNC1 in the NPC cell line 5-8F. PHB was found to be upregulated when LPLUNC1 was overexpressed. Specifically, the expression of PHB was examined with an NPC tissue microarray (TMA) using immunohistochemical analysis, which was further verified using quantitative reverse transcriptase (qRT)-PCR in pure NPC epithelium tissues. Subsequently, correlations between the expression of PHB with various clinicopathological characteristics of NPC were analysed. We found that PHB expression levels were significantly downregulated in NPC patients and that low PHB expression patterns paralleled the carcinogenetic process of NPC, which was closely related to poor patient survival. These findings suggest that PHB plays a role in tumour suppression in NPC and that it might be a new biomarker for NPC.
Materials and methods
Cell culture and stable transfection
The 5-8F cell line (an NPC cell line with high tumorigenic and metastatic ability) was kindly provided by the Cancer Centre of Sun Yet-Sen University (Guangzhou, People’s Republic of China). Cell transfection was carried out using Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen, Carlsbad, California, USA). A total of 5×104 5-8F cells were plated into each well of a 12-well plate 24 h before transfection. For each transfection, 2 μg of empty pIRESneo3 vector (Clontech, Palo Alto, California, USA) or LPLUNC1 pIRESneo3 (a full-length LPLUNC1 cDNA introduced into the pIRESneo3 vector) was transfected into 5-8F cells (designated as 5-8F/Vector and 5-8F/LPLUNC1 cells, respectively). The cells were incubated at 37°C for 5 h and then the transfection media were replaced with fresh complete culture media. Stably transfected cell lines were selected using geneticin (G418, 200 µg/ml; Invitrogen). The expression of LPLUNC1 was confirmed by qRT-PCR and western blot analysis.
Two-dimensional fluorescence difference gel electrophoresis, in-gel digestion and protein identification
The LPLUNC1-induced differential expression of proteins in NPC cells was characterized by 2-D DIGE analysis as described previously (Cilia et al., 2009; Wang et al., 2011). Briefly, 5-8F/Vector and 5-8F/LPLUNC1 cells were harvested, and protein lysates were extracted. Subsequently, the lysates were treated using the ReadyPrep 2D Clean-up kit according to the manufacturer’s instructions (Bio-Rad, Hercules, California, USA). The lysates were resuspended in lysis buffer compatible with DIGE labelling (GE Healthcare, Piscataway, New Jersey, USA) and analysed for protein concentration using BCA (Pierce, Beijing, People’s Republic of China). The protein lysates were subjected to fluorescence dye labelling at a dye/protein ratio of 400 pmol/100 µg for 30 min. Then, 20 µg of proteins from each group were mixed with the same volume of DIGE 2× buffer (8 mol/l urea, 4% w/v CHAPS, 2% w/v DTT, 2% v/v pharmalytes, pH 3–10 for IEF). In addition, 20 µg of individual samples were diluted in the rehydration solution (8 mol/l urea, 0.5% w/v CHAPS, 0.2% w/v DTT, 0.2% v/v pharmalyte, pH 3–10). Samples were loaded onto IPG strips (18 cm, pH 3–10, nonlinear, GE Healthcare) for 2-D gel electrophoresis. Fluorescence images were acquired using the Ettan DIGE imager (GE Healthcare), and DIGE gels were analysed using the DIA module of the DeCyder software (version 6.5; GE Healthcare).
To prepare gels to capture the spots of interest, 500–1000 µg of protein was subjected to 2-D DIGE on IPG strips and stained with Coomassie brilliant blue. The protein spots of interest were excised and destained with 25 mmol/l ammonium bicarbonate/50% acetonitrile (CAN), and in-gel digestion was performed with 0.01 µg/µl trypsin (Promega, Madison, Wisconsin, USA) in 25 mmol/l ammonium bicarbonate for 15 h at 37 °C. The hydrolysates were collected, and the tryptic peptides were extracted from the gel pieces sequentially with 5% TFA at 40°C for 1 h and then 2.5% TFA/50% ACN at 30°C for 1 h. The extracts were pooled, lyophilized and stored at −20°C until use. Gel pieces from a ‘blank’ region and from the BSA molecular mass marker were used as negative and positive controls, respectively.
The peptide mixtures were redissolved in 0.5% TFA, and 1 µl of peptide solution was mixed with an equal volume of matrix (4-hydroxy-α-cyanocinnamic acid in 30% ACN/0.1% TFA). Then, the peptides were spotted on the target plate. Individual protein peptides were identified by MALDI–TOF mass spectrometry on a 4700 Proteomics Analyser (Applied Biosystems, Foster City, California, USA). The mass spectra were used to examine human protein sequences in the Swiss-Prot database using the Mascot database search algorithm (version 1.9).
For the mRNA expression study, we used 24 NPC tissues and eight normal nasopharyngeal epithelium (NPE) samples from biopsy-negative cases as controls. Both sets of samples and information on their pathological diagnosis were obtained from patients in the Second Xiangya Hospital (Changsha, People’s Republic of China) and Hunan Province Tumour Hospital in 2010. We used a LEICA CM 1900 (Leica, Solms, Germany) for frozen sections and the Leica AS LMD system (Leica) to obtain the pure tissues.
To prepare the NPC TMA, we used 323 patients with NPC and NPE. The records for the 323 patients, including name, sex, age, pathological diagnosis, tumour, nodal status, metastasis and tumour-node-metastasis classification, were collected at the Ear, Nose and Throat (ENT) Department at Xiangya Hospital (Changsha, People’s Republic of China) from January 2002 to October 2004. The clinicopathological information is presented in Table 1.
All the samples were fully encoded and examined under a protocol approved by the Institutional Review Board of Human Subjects Research Ethics Committee. All of the individuals participating in this project signed an informed consent. Data on clinical outcome were obtained from the patients’ records.
RNA isolation and real-time RT-PCR
Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. RNA was extracted with RNeasy and treated with DNase I according to the manufacturer’s instructions (Qiagen, Valencia, California, USA). The integrity and quality of RNA were confirmed using agarose gel electrophoresis and detection of absorbance of 260 nm. Total RNA was reverse transcribed to cDNA using the SuperScript First-Strand Synthesis System with random hexamer primers (Promega).
Real-time qRT-PCR was performed using a cDNA template and the following primer pairs: PHB 5′-TCCAGGCAGGTGAGCGACGA-3′ (forward) and 5′-GCGCAG CTCGATCAGGCCAT-3′ (reverse); GAPDH 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse). All reactions were performed in triplicate with expression levels normalized against GAPDH.
The TMA was constructed as described in our previous study (Fan et al., 2006). IHC studies were performed using the standard streptavidin/peroxidase staining method as described previously (Zhang et al., 2009). For immunohistochemical staining, 5-μm sections from each paraffin block were stained with antibodies against rabbit anti-PHB at a dilution of 1 : 400 (E-Star, Shanghai, People’s Republic of China). All known positive sections were taken as positive controls. Negative mouse serum and PBS were used instead of the primary antibody as a negative control and blank control, respectively. A semiquantitative scoring criterion for IHC was used in which the staining intensity and positive areas were recorded. The intensity of PHB was scored as follows: an IHC score of 0 indicated a negative expression, and 1, 2 and 3 indicated weak, medium and strong positive expression, respectively. For the statistical analysis, the two scores were combined to obtain the final score: negative (IHC score 0–1) or positive (IHC score 2–3).
Because some NPC patients could not be contacted for follow-up and some tissue sections were unusable because tissue sections were missing, the survival data were available for 84 patients. The median follow-up time was 45 months (3–75 months). Overall survival (OS) was defined as the time of diagnosis to the date of death. Progression-free survival (PFS) was defined as the time of diagnosis to the date of first failure. The OS and PFS estimates over time were calculated using the Kaplan–Meier method, and differences were compared using the log-rank test. Cox regression model analysis was used for the analysis of factors potentially related to PFS or OS. The results of the analysis were considered significant in a log-rank test if P value was less than 0.05.
Statistical analyses were carried out in SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA). The χ2-test was used to determine whether two groups had distinct gene expression levels; a P value of less than 0.05 was considered statistically significant.
Identification of differentially expressed proteins as regulated by LPLUNC1 through MALDI–TOF/TOF in NPC
As the LPLUNC1 gene is a nasopharyngeal epithelium tissue-specific gene and is downregulated in NPC, we aimed to explore the downstream targets of the LPLUNC1 gene in NPC cells. We constructed a LPLUNC1-overexpressing NPC cell line through the introduction of the LPLUNC1 gene into 5-8F cells through stable transfection. As shown in Fig. 1, the expression of LPLUNC1 was confirmed by qRT-PCR and western blot analysis.
The total cell extract of 5-8F/LPLUNC1 and 5-8F/Vector cells was separated by 2-D DIGE. Next, the Cy2, Cy3 and Cy5 channels of individual gels were imaged and analysed using the DeCyder 5.0 software. A protein feature of 30 kDa and pI 5.57 was significantly increased in 5-8F/LPLUNC1 cells (Fig. 2). This protein was identified as PHB using MALDI–TOF/TOF (Fig. 3).
Downregulation of PHB mRNA in NPC
PHB is a critical regulator of cell proliferation and cycling and is associated with tumorigenesis. Thus, we explored the expression of PHB in a clinical setting. Specifically, eight NPE specimens and 24 NPC specimens were obtained to verify the PHB expression in these tissues using qRT-PCR. All tissue specimens were purified using laser-capture microdissection before RNA extraction. Figure 4 shows that the average expression levels of PHB were lower in the NPC specimens than in the NPE tissues (P<0.001).
Correlation of PHB expression with the clinicopathological characteristics of NPC
The status of PHB expression was examined with a TMA using immunohistochemical analysis. Sections were evaluated independently three times by a pathologist blinded to outcome data. Stained sections of TMA were graded for their cytoplasmic IHC staining intensity (IHC score) against the level of PHB protein from 0 (no staining) to 3 (strong staining intensity) (Fig. 5). High (IHC score 2–3, 60.3%) expression was observed in NPE tissues, whereas low or negative (IHC score 0–1, 80.2%) expression was observed in NPC tissues. The expression levels of PHB and various clinicopathological characteristics are listed in Table 2. As expected, the levels of PHB expression were significantly downregulated in NPC tissues (χ2=18.344, P<0.001), and the decreased PHB expression appeared to be associated with advanced clinical stage (P=0.031) and metastasis (P=0.002) of NPC.
Negative PHB expression is associated with shorter survival
Next, we examined the prognostic value of PHB expression. The Kaplan–Meier survival analyses showed that NPC patients with negative expression levels of PHB had a significantly shorter PFS (P=0.001) and OS (P=0.002) than those with positive levels of PHB (Fig. 6). These findings suggest that the expression level of PHB can serve as an important and independent predictor in NPC patients. Furthermore, multivariate Cox regression analysis of various parameters with PFS and OS showed that the negative expression of PHB was a significant predictive factor for poor outcomes (PFS with hazard ratios=0.410, P=0.016, 95% confidence interval=0.198–0.847; OS with hazard ratios=0.417, P=0.018, 95% confidence interval=0.202–0.862); however, there were no differences among the groups in terms of sex, age, clinical stage and metastasis.
NPC is a malignant disease that has critically threatened human health. NPC is typically diagnosed at advanced clinical stages, resulting in poor outcomes. Thus, it is important to explore the potential biomarkers that may be related to early diagnosis and prognosis of NPC. Utilizing a series of large-scale screening techniques from proteomics to TMA, PHB has been preliminarily verified as a molecular marker in NPC.
PHB expression was upregulated in response to LPLUNC1 overexpression, as detected using 2-D DIGE. LPLUNC1 is a member of the PLUNC family. Our earlier studies have indicated that palate, lung and nasal epithelium clone (PLUNC) genes might be putative molecular markers of NPC (Zhou and Zeng 2008). Thus, there may be a close relationship between PHB and NPC, and we speculated that the PHB gene might play an important role in the occurrence and development of NPC. PHB is ubiquitously expressed in cells. Several studies suggest that PHB may suppress tumour growth by regulating transcription and transforming growth factor-β signalling (Rizwani et al., 2009; Zhu et al., 2010). PHB co-localizes with and binds to p53 to increase p53 transcriptional activity (Fusaro et al., 2003; Chander et al., 2010). PHB levels are decreased in gliomas (Chumbalkar et al., 2005), gastric adenocarcinoma (Liu et al., 2009), prostate cancer (Dart et al., 2009), ovarian cancer (Dai et al., 2010) and liver cancer (Ko et al., 2010). Conversely, in large studies examining oesophagus (Ren et al., 2010), stomach (Kang et al., 2008), thyroid (Gu et al., 2010) and breast carcinomas (Gregory-Bass et al., 2008; He et al., 2010), PHB levels were increased. Considering the current evidence, the role of PHB expression in different tumours is unclear. In this study, we examined the relationship between alterations in PHB expression and the prognosis of patients with NPC.
Therefore, we used qRT-PCR to examine the expression of PHB and found that NPC tissues had a significantly lower PHB expression than the paired NPE tissues at mRNA level. The additional IHC study showed that the decrease in the PHB protein expression level corresponded to the degree of differentiation of NPC; a significant decrease was found in high-grade NPC tissues compared with low-grade cancer tissues. In addition, low levels of PHB protein expression were closely associated with metastasis in NPC lesions. Thus, low expression levels of PHB might play an important role in the progression of NPC.
An analysis of survival data indicated that the downregulated expression of PHB was significantly associated with a poor prognosis of NPC. In addition, with a multivariate Cox regression analysis, we showed that the negative expression of PHB was a significant predictive factor for a poor prognosis in NPC patients. These results also suggest that PHB immunohistochemical staining might provide clinically useful prognostic information in cases of NPC.
Our results showed that the expression level of PHB was significantly downregulated in NPC tissues irrespective of the mRNA and protein level compared with NPE tissues. Furthermore, low expression levels of PHB were significantly correlated with the progression of NPC and shorter survival of patients. These results indicate that PHB may be a useful indicator for prognosis and survival of patients with NPC and that it might provide a basis for the development of a novel biomarker for diagnosis and prognosis of NPC. However, further investigations involving additional NPC patients as well as studies of the function of PHB in NPC need to be performed in vivo and in vitro.
The authors are grateful to the Ear, Nose and Throat (ENT) Department at Xiangya Hospital for providing NPC samples. This work was supported by grants from the following sources: the National Science Foundation of China (81171930, 81071644, 81172189, 81101509); the 111 project (111-2-12); and the Program for New Century Excellent Talents in University (NCET-08-0562).
Conflicts of interest
There are no conflicts of interest.
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