NM23 expression is closely associated with hepatocellular carcinoma (HCC) recurrence, but the hereditary factors influencing NM23 levels are unknown. Using public database, the diagnostic value of
Primary liver cancer (PLC) is a common malignant neoplasm, with an estimated 854 000 incident cases and 810 000 deaths globally in 2015, contributing to 20 578 000 disability–adjusted life-years [
NME/NM23 nucleoside diphosphate kinase 1 (
Recently, genome–wide association studies (GWAS) have become an efficient method to study the molecular genetics of HCC development and progression [
The NM23 specific isoform expression in HCC was obtained from GEO (http://www.ncbi.nlm.nih.gov/geo/) and Oncomine (https://www.oncomine.com/). The criteria used to determine study eligibility were as follows: (1) human species; (2) histopathology confirmed with HCC; (3) availability NME1 expression in HCC and paracancerous; (4) use of prospective or retrospective cohort design with a clearly defined source population and justify all excluded eligible cases; and (5) selection of the latest and most complete study to avoid duplication. NM23 expression in tumor and nontumor tissues was presented as mean and standard deviation (SD) and compared by Student’s test. The receiver operating characteristic (ROC) curve was performed to identify the diagnostic value of
A total of 424 patients were enrolled at the First Affiliated Hospital of Guangxi Medical University (Guangxi, China) from 2005 to 2013. All HBV-related HCC subjects were histopathologically confirmed after hepatectomy. This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University.
All tumor tissues were immunohistochemically stained for NME1 by full–time pathologists according to routine processes and the guidelines for standardized pathological diagnosis of primary liver cancer. Supersensitive reagents and mouse anti-human NME1 monoclonal antibody (clone OTI4G3) and streptavidin-peroxidase anti-human kit were purchased from OriGene (Beijing OriGene Technologies, Inc., China). The immunohistochemical staining was carried out by following the manufacturer’s instructions of the kit. For the negative control, the primary antibody was replaced by phosphatebuffered saline/Tween. Positive staining for the NM23 protein appeared as yellow–brown particles.
The criteria used to analyze NM23 expression were based on the number and staining intensity of the stained cells [
(a) Representative staining of NM23 protein (negative, weak, moderate, and strong) in HCC tissues. Original magnifications were ×100 and ×400, respectively. (b) The flowchart depicts the process of the GWAS, screening the candidate SNPs, and association analysis in this study. (c) Principal components analysis (PCA) for ancestry and population stratification implemented in the EIGENSOFT package. The blue dots represent negative NM23 expression, the red dots represent weak NM23 expression, the green dots represent moderate NM23 expression, and the blue dots represent strong NM23 expression. (d) Quantile–Quantile (Q–Q) plot for the GWAS results. The genomic inflation factor was calculated by MATLAB 7.0 based on the
DNA was extracted from HCC specimens (including paracancer tissues) using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. DNA yield and purity were measured by the NanoDrop2000 system (Thermo Fisher Scientific, Waktham, MA, USA).
We genotyped all samples on the Illumina Human Exome BeadChip-12-1_A, which includes 242,901 markers focused on protein-altering variants. Genotype calling was carried out using Genotyping Module v1.0 in GenomeStudio. A total of 50 samples (over 10%) were randomly selected using random number table and sequenced for candidate loci by ABI Prism 3100 (Applied Biosystems, Shanghai Sangon Biological Engineering Technology & Services, Shanghai, China), yielding a 100% concordance rate with the genotyped variants (the SNP Sequenced primers were showed in Supplementary Table
All patients were followed up after discharge until death or the last time of follow-up (September 2014) by personal or family contacts. The median follow-up time for the 424 patients was 42 months (ranging from 4 to 125 months), and the median survival time (MST) was 30 months. Overall survival (OS) and recurrence-free survival (RFS) were calculated.
We performed a GWAS following the process displayed in the flowchart (Figure
Population stratification was estimated by a principal components analysis (PCA), implemented by the EPACTS package in MATLAB 7.0 (MathWorks, Natick, MA, USA). A quantile–quantile (Q–Q) plot was used to evaluate the potential impact of population stratification. QC filtering was performed with PLINK version 1.07, R v. 3.0.1 (
A linear regression model was used to test the association of quantitative trait (intensity of NM23 expression) with the SNPs that passed QC using the EPACTS package version 3.2.6 [
We selected all genes containing candidate SNPs to search for gene–gene interactions. The signaling pathway network diagram was performed with GeneMANIA Software [
The Kaplan–Meier lifetable method was used to calculate OS and RFS rates, and differences in survival rates were estimated using a generalized log-rank test. The association between candidate gene and clinical outcomes of HCC patients was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) [
A total of 28 datasets were enrolled in this study from GEO and Oncomine (Supplementary Table
The NM23 expression of HCC and nontumor tissues in public datasets.
The ROC analysis of NM23 expression in public HCC and nontumor tissues datasets. Blue: identity cure; red: sensitivity cure for the differentiation of HCC from nontumor tissues.
The groups were similar with respect to most of the characteristics (
Clinicopathological characteristics of HBV-related HCC cases after data quality control.
NM23 | OS | RFS | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
— | 1+ | 2+ | 3+ |
|
|
Total |
MST |
|
|
MRT |
|
|
|
Gender | 0.281 | 0.400 | 0.651 | ||||||||||
Male | 40 | 241 | 39 | 45 | 1.01 |
365 | 48 | 0.93 |
7 | 1.09 |
|||
Female | 3 | 31 | 7 | 2 | Ref. | 43 | 82 | Ref. | 12 | Ref. | |||
Age (years) |
|
0.485 | 0.718 | ||||||||||
≤46 | 20 | 144 | 35 | 24 | 1.45 |
223 | 61 | 0.87 |
7 | 1.05 |
|||
>46 | 23 | 128 | 11 | 23 | Ref. | 185 | 45 | Ref. | 11 | Ref. | |||
Race | 0.159 | 0.972 | 0.761 | ||||||||||
Han | 32 | 174 | 25 | 26 |
|
257 | 48 | 0.92 |
7 | 0.96 |
|||
Minority | 11 | 98 | 21 | 21 | Ref. | 151 | 50 | Ref. | 11 | Ref. | |||
Preoperation TACE | 0.359 | 0.640 | 0.592 | ||||||||||
No | 30 | 215 | 36 | 40 | 1.46 |
321 | 51 | 0.91 |
10 | 0.92 |
|||
Yes | 13 | 57 | 10 | 7 | Ref. | 87 | 44 | Ref. | 6 | Ref. | |||
Postoperation TACE | 0.530 | 0.717 | 0.416 | ||||||||||
No | 26 | 156 | 25 | 22 | 0.76 |
229 | 47 | 0.94 |
7 | 0.90 |
|||
Yes | 17 | 116 | 21 | 25 | Ref. | 179 | 76 | Ref. | 13 | Ref. | |||
Smoking status | 0.967 | 0.088 | 0.053 | ||||||||||
None | 14 | 99 | 16 | 17 | 1.04 |
146 | 39 | 1.24 |
5 | 1.31 |
|||
Ever | 29 | 173 | 30 | 30 | Ref. | 262 | 61 | Ref. | 12 | Ref. | |||
Drinking status | 0.471 | 0.359 | 0.443 | ||||||||||
None | 16 | 107 | 19 | 24 | 1.32 |
166 | 44 | 0.82 |
6 | 1.11 |
|||
Ever | 27 | 165 | 27 | 23 | Ref. | 242 | 51 | Ref. | 11 | Ref. | |||
BMI |
|
0.480 | 0.603 | ||||||||||
≤25 | 35 | 206 | 39 | 45 |
|
325 | 48 | 1.03 |
11 | 0.92 |
|||
>25 | 8 | 66 | 7 | 2 | Ref. | 83 | 63 | Ref. | 6 | Ref. | |||
serum AFP (ng/ml) | 0.099 | 0.101 | 0.353 | ||||||||||
≤400 | 23 | 143 | 19 | 19 |
|
204 | 61 | 1.00 |
13 | 0.88 |
|||
>400 | 16 | 108 | 26 | 26 | Ref. | 176 | 41 | Ref. | 6 | Ref. | |||
NA | 4 | 21 | 1 | 2 | 28 | ||||||||
Child–Pugh | 0.509 |
|
|
||||||||||
A | 35 | 237 | 37 | 39 | 0.88 |
348 | 51 | 0.76 |
9 | 0.66 |
|||
B | 8 | 35 | 9 | 8 | Ref. | 60 | 31 | Ref. | 7 | Ref. | |||
BCLC stage | 0.183 |
|
|
||||||||||
A | 30 | 153 | 26 | 22 | 0.66 |
231 | 101 | 1.49 |
14 |
|
|||
B | 4 | 46 | 10 | 6 | 0.92 |
66 | 39 | 1.92 |
6 | 0.66 |
|||
C | 9 | 73 | 10 | 19 | Ref. | 111 | 27 | Ref. | 3 | Ref. | |||
Number of tumors | 0.247 |
|
0.137 | ||||||||||
Single(n=1) | 36 | 202 | 32 | 31 | 0.63 |
301 | 58 | 0.71 |
11 | 0.80 |
|||
Multiple(n>1) | 7 | 70 | 14 | 16 | Ref. | 107 | 28 | Ref. | 5 | Ref. | |||
Tumor size (cm) | 0.659 |
|
0.175 | ||||||||||
≤10 | 37 | 222 | 36 | 36 | 0.72 |
331 | 51 | 0.66 |
12 | 0.81 |
|||
>10 | 6 | 50 | 10 | 11 | Ref. | 77 | 40 | Ref. | 4 | Ref. | |||
Intrahepatic metastasis | 0.325 |
|
|
||||||||||
Absence | 24 | 147 | 26 | 19 | 0.79 |
216 | 76 | 1.01 |
13 |
|
|||
Presence | 19 | 125 | 20 | 28 | Ref. | 192 | 36 | Ref. | 4 | Ref. | |||
Distant metastasis | 0.586# | 0.051 | 0.178 | ||||||||||
Absence | 40 | 249 | 40 | 41 | 0.64 |
370 | 51 | 0.38 |
11 | 0.77 |
|||
Presence | 3 | 23 | 6 | 6 | Ref. | 38 | 32 | Ref. | 3 | Ref. | |||
Cirrhosis | 0.246 | 0.567 | 0.923 | ||||||||||
Absence | 6 | 34 | 3 | 2 | 0.54 |
45 | 48 | 0.99 |
13 | 0.98 |
|||
Presence | 37 | 237 | 43 | 45 | Ref. | 362 | 48 | Ref. | 7 | Ref. | |||
NA | 0 | 1 | 0 | 0 | 1 | ||||||||
Pathological grade | 0.571 | 0.267 | 0.105 | ||||||||||
Well | 4 | 17 | 2 | 1 | 0.54 |
24 | 39 | 1.07 |
4 | 1.80 |
|||
Moderately and poorly | 35 | 222 | 39 | 36 | Ref. | 332 | 47 | Ref. | 7 | Ref. | |||
NA | 4 | 33 | 5 | 10 | 52 | ||||||||
Regional invasion |
|
0.407 | 0.215 | ||||||||||
Absence | 38 | 240 | 34 | 34 |
|
346 | 51 | 0.81 |
11 | 0.81 |
|||
Presence | 5 | 32 | 12 | 13 | Ref. | 62 | 40 | Ref. | 3 | Ref. | |||
PVTT | 0.162 |
|
|
||||||||||
None | 37 | 225 | 42 | 32 | 0.35 |
336 | 74 |
|
12 |
|
|||
VP1 | 1 | 9 | 0 | 1 | 0.24 |
11 | 28 |
|
2 | 0.37 |
|||
VP2 | 0 | 11 | 1 | 5 | 0.93 |
17 | 18 | 0.53 |
2 | 0.64 |
|||
VP3 | 4 | 23 | 2 | 6 | 0.39 |
35 | 18 | 0.36 |
3 | 0.49 |
|||
VP4 | 1 | 4 | 1 | 3 | Ref. | 9 | 8 | Ref. | 2 | Ref. | |||
Radical resection | 0.847 |
|
0.306 | ||||||||||
No | 24 | 147 | 29 | 28 | 1.14 |
228 | 73 | 1.01 |
12 | 0.87 |
|||
Yes | 18 | 114 | 17 | 19 | Ref. | 168 | 40 | Ref. | 6 | Ref. | |||
NA | 1 | 11 | 0 | 0 | 12 | ||||||||
Antiviral therapies |
|
|
0.800 | ||||||||||
No | 36 | 174 | 26 | 28 |
|
264 | 41 |
|
6 | 1.03 |
|||
Yes | 7 | 98 | 20 | 19 | Ref. | 144 | NA (>45) | Ref. | 13 | Ref. | |||
NM23 expression | NA | 0.378 | 0.802 | ||||||||||
Negative | 43 | - | - | - | 43 | 47 | 1.12 |
14 | 0.88 |
||||
1+ | - | 272 | - | - | 272 | 45 | 1.09 |
7 | 1.08 |
||||
2+ | - | - | 46 | - | 46 | 73 | 0.68 |
7 | 0.99 |
||||
3+ | - | - | - | 47 | 47 | 40 | Ref. | 11 | Ref. |
A total of 408 HBV-related HCC patients and 21,529 SNPs were included in further analysis after QC filtering. A PCA plot demonstrated that there were no outliers in this study population (Figure
Based on the results of the GWAS displayed in a Manhattan plot (Figure
The signaling pathway network showed that
The association of
Haplotype analysis revealed that six SNPs of
Association between haplotypes of
Haplotypes | NM23 | Ad OR★ |
|
Ad |
|
Ad |
|
|||
---|---|---|---|---|---|---|---|---|---|---|
— | 1+ | 2+ | 3+ | (95% CI) | (95% CI) | (95% CI) | ||||
PSORS1C1 | ||||||||||
TGCACA | 40 | 273 | 61 | 63 | Ref. | Ref. | Ref. | |||
CAGGTG | 37 | 171 | 19 | 20 | 0.44 |
|
0.71 |
0.219 | 0.87 |
0.653 |
CAGACA | 6 | 64 | 9 | 5 | 0.56 |
|
0.99 |
0.967 | 1.66 |
0.073 |
Others |
3 | 36 | 3 | 6 | 0.60 |
0.135 | 0.97 |
0.805 | 2.04 |
0.054 |
All others |
46 | 271 | 31 | 31 | 0.48 |
|
0.93 |
0.561 | 0.74 |
0.136 |
STARD3 | ||||||||||
GG | 46 | 329 | 58 | 69 | Ref. | Ref. | ||||
Others |
40 | 209 | 34 | 25 | 0.65 |
|
0.97 |
0.814 | 1.18 |
0.498 |
The eQTL mapping was performed in a total number of 153 samples in GTEx portal. Rs1877031 and rs11869286 presented a negative eQTL relationship in liver samples (both normalized effect size=-0.25,
MST was calculated using the Kaplan–Meier method with a log-rank test in the different subgroups of baseline variables (Table
The prognostic value of
Kaplan–Meier survival analysis of
After hepatic resection, there was a significant difference in the MST of
Kaplan–Meier survival analysis of SNPs in
A Cox proportional hazard regression model was applied to perform stratified analysis and further assessed the relationship between the SNPs with OS. We found that
The association between
In this study, we investigated the diagnostic value of
HCC is the third leading cause of cancer-related deaths worldwide [
A correlation between
Several limitations of this study warrant discussion. First, our sample size is modest, as is common to many pharmacogenomic GWAS, and additional studies with larger sample sizes and multiple centers are needed to clarify our results. In addition, because the subjects evaluated in this study included minority subjects, racial heterogeneity may also represent a major limitation of the study. However, we accounted for this by including race, age, and gender as covariates in our GWAS model, and based on the low genomic inflation factor and the Q–Q plot, there is no evidence of population stratification. Finally, our research is preliminary, and further mechanistic and functional studies should be undertaken to discern the potential role of variants near
In summary, we identified high NM23 mRNA level offering high diagnostic ability for the discrimination of HCC and demonstrated that genetic variants near
All the data supporting our findings can be found in the Results and Supplementary materials section of the paper. Please contact corresponding author for more data on reasonable request.
The authors declare no conflicts of interest.
This work was supported in part by the National Nature Science Foundation of China (Grant nos. 81560535, 81072321, 30760243, 30460143, and 30560133), 2009 Program for New Century Excellent Talents in University (NCET), Guangxi Nature Sciences Foundation (no. GuiKeGong 1104003A-7), and Guangxi Health Ministry Medicine Grant (Key-Scientific Research–Grant Z201018). The authors would also like to acknowledge the support by the National Key Clinical Specialty Programs (General Surgery & Oncology) and the Key Laboratory of Early Prevention & Treatment for Regional High–Incidence–Tumor (Guangxi Medical University), Ministry of Education, China. Thanks are also due to TCGA group, GTEx, GEO, and Oncomine that database provided relevant data.