Oncomine database analysis
To analyze the expression levels of specific ECRGs in a variety of malignancies, Oncomine (http://www.oncomine.org) was used, which is an online cancer microarray database including 715 datasets and 86,733 samples to facilitate and promote discovery from genome-wide expression analyses [20]. Paired Student’s t-test was used. A fold-change of 2 with P<0.01 was defined as clinically significant.
Gene Set Cancer Analysis (GSCA) database analysis
Gene Set Cancer Analysis (GSCA) is an integrated genomic and immunogenomic web-based platform for gene set cancer research [21]. In this study, gene expression levels of ECRG factors across multiple cancer types were calculated using the GSCA online database (http://bioinfo.life.hust.edu.cn/GSCA/).
Cancer Cell Line Encyclopedia (CCLE) database analysis
The mRNA levels of distinct ECRG factors in multiple types of cancer cell lines were determined using the CCLE database (http://portals.broadinstitute.org/ccle), which is an online encyclopedia of a data collection of gene expression, copy numbers and massively parallel sequences from 1,457 human cancer cell lines [22].
cBioPortal cancer genomics database analysis
The impact of genomic alterations of ECRGs containing gene mutations and copy-number variance on the overall survival (OS) and disease-free survival (DFS) rates of patients with HCC were calculated using the cBioPortal online database (www.cbioportal.org) [23]. cBioPortal for Cancer Genomics allows the visualization, analysis and download of large-scale cancer genomics datasets.
Kaplan-Meier Plotter survival analysis
The prognostic impact of ECRG mRNA expression were analyzed using the Kaplan-Meier Plotter online database (http://kmplot.com/analysis/), which is capable of assessing the effect of 54k genes (mRNA, miRNA, protein) on survival in 21 cancer types including HCC [24, 25]. To investigate the overall survival (OS), relapse-free survival (RFS) and progression-free survival (PFS) in patients with HCC, the clinical samples were divided into high and low-expression groups by the median value of mRNA expression. After the target probe was separately entered into the database, survival analyses were carried out to generate Kaplan-Meier plots.
Tissue specimens
A tissue microarray chip was purchase from Outdo Biotech Co., Ltd. (Shanghai, China) for immunohistochemistry, providing 100 primary HCC tissues along with 80 paired adjacent noncancerous tissues from patients undergoing curative surgery between February 2008 and June 2010. The median postoperative follow-up period was 41 months (range, 1-88 months). During the follow-up period, 40 (40.8%) patients had died because of disease recurrence and distant metastasis. Tumor grade and stage were classified in accordance with the International Union against Cancer (UICC)/American Joint Committee on Cancer (AJCC) pathologic tumor‑node‑metastasis (TNM) classification, 7th edition (2010). The clinicopathological parameters are summarized in Table 1. All cases were confirmed by two pathologists. No patients had received neoadjuvant therapy before surgery. Signed informed consents were obtained from patients in accordance with the principles expressed in the Declaration of Helsinki. This study was approved by the Institution Review Board of People’s Hospital of Ningxia Hui Autonomous Region.
Table 1
Correlation between ECRG4 expression and clinicopathological factors in HCC patients
Parameters
|
ECRG4 expression
|
χ2
|
P value
|
Low, n (%)
|
High, n (%)
|
Age (years)
|
|
|
|
|
<68
|
20
|
36
|
0.332
|
0.565
|
≥68
|
17
|
24
|
|
|
Sex
|
|
|
|
|
Female
|
16
|
24
|
0.099
|
0.753
|
Male
|
21
|
36
|
|
|
Histological grade
|
|
|
|
|
I/II
|
21
|
58
|
21.644
|
<0.001
|
III
|
16
|
3
|
|
|
T classification
|
|
|
|
|
T2
|
4
|
0
|
11.455
|
0.003
|
T3
|
17
|
45
|
|
|
T4
|
16
|
16
|
|
|
N classification
|
|
|
|
|
N0
|
11
|
40
|
11.856
|
0.013
|
N1/N2
|
26
|
21
|
|
|
M classification
|
|
|
|
|
M0
|
32
|
61
|
1.611
|
0.204
|
M1
|
5
|
0
|
|
|
Clinical stage (pTNM)
|
|
|
|
|
I/II
|
10
|
40
|
13.694
|
<0.001
|
III/IV
|
27
|
21
|
|
|
Immunohistochemistry and evaluation
Immunohistochemical analysis for ECRG4 was performed using the standard EnVision complex method as described previously [19]. 4-µm sections were cut from specimens that had been fixed in 10% buffered formalin and embedded in paraffin. After undergoing deparaffinization (xylene, 2 times and 10 min each at 37˚C) and rehydration (alcohol gradient, 100, 95, 80 and 70%), endogenous peroxidase blocking (0.3% hydrogen peroxide, 30 min at room temperature) and antigen retrieval (at 121˚C in citrate buffer at 10 mM, pH 6.0 for 10 min), specimens were incubated with a rabbit polyclonal anti-ECRG4 antibody (catalog no. PA5-38791; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a dilution of 1:400 overnight at 4˚C. Immunohistochemical staining was conducted by an EnVision antibody complex (anti-mouse/rabbit) method in conjunction with an EnvisionTM Detection kit (ZSGB-BIO, Beijing, China) with 3,3′-diaminobenzidine as the chromogen substrate. Nuclei were counterstained with 0.5% hematoxylin for 2 min at room temperature. Sections immunostained with normal rabbit IgG (catalog no. ab188776; dilution, 1:50; Abcam, Cambridge, MA, USA) as the primary antibody were used as negative controls. The staining evaluation was performed as follows: ten random 400× microscopic fields per slide were evaluated by two independent observers who were blinded to the clinical information. Global ECRG4 immunostaining was scored using a semi-quantitative integration method combining the percentage of positive cells and the staining intensity. The mean percentage of positively stained cells was scored as follows: 0% (0); 1%-25% (1); 26%-50% (2); 51%-75% (3); and 76%-100% (4). The staining intensity was categorized as follows: absent (0); weak staining exhibited as light yellow (1); moderate staining exhibited as yellow brown (2); and strong staining exhibited as brown (3). The multiplication of the intensity and extent scores was used as the final staining score. For the purpose of statistical evaluation, tumors having a final staining score of ≤3 were designated as low expression and those with scores >3 as high expression.
Statistical analyses
All statistical analyses were performed using the SPSS 17.0 statistical software package (SPSS Inc, Chicago, IL, USA). Associations between different expression levels of ECRG factors and clinicopathological features were analyzed using a χ2 test. For Kaplan-Meier Plotter and prognostic relevance of ECRG mRNA levels in HCC patients, survival was estimated according to the Kaplan-Meier method and the log-rank test. Significant factors were identified by univariate analysis, and further examined by multivariate regression analysis with a Cox proportional-hazards regression model. P<0.05 defined statistical significance.