Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and is the third leading cause of cancer-related death due to its extremely poor prognosis [
MicroRNA (miRNA) is a class of single-stranded, 19–25 nucleotides in length, noncoding RNA molecules in eukaryotes. Binding to the 3′-untranslated region (3′-UTR) of target mRNAs, miRNA regulates the translation and degradation of its target mRNAs and thus influences the gene expression [
Single-nucleotide polymorphisms (SNPs), one form of DNA variation, are pervasive in miRNAs, including pri-miRNAs, pre-miRNAs, and mature miRNAs. They could influence various biological processes by changing the secondary structure of pre-miRNAs, interfering the maturation and/or target selection of miRNAs [
Previous studies have shown that miR-646 is implicated in human cancers [
Subjects were recruited from a case-control Chinese population as described previously [
Genomic DNA was isolated from the peripheral blood of the participants using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, USA). Genotyping was done using Sequenom MassARRAY technique. The amplification primers were 5′-ACGTTGGATGCACACCTGCTTTTCACCTGT-3′ and 5′-ACGTTGGATGGTAAAGATAGGCCACTGAGC-3′, while the extension primer was 5′-CCCCCAGGAAGCAGCTGCCTC-3′.
Statistical analyses were performed using SPSS (version 13.0) and Excel. Observed and expected genotype frequencies were evaluated for Hardy-Weinberg equilibrium (HWE) using Pearson’s chi-square test. Odds ratio (OR) adjusted for age, gender, smoking status, and drinking status, along with 95% confidence interval (CI), was determined by logistic regression. Pearson’s chi-square test was also used to assess qualitative data among different groups, while Student’s
997 HCC patients (including 771 HBV-related HCC patients) and 993 controls were enrolled in this study. The demographic characteristics were summarized in Table
General characteristics in hepatocellular carcinoma patients and controls.
Cases ( |
Controls ( |
|
|
---|---|---|---|
Number (%) or mean ± SD | Number (%) or mean ± SD | ||
Age (years) | 54.70 ± 11.27 | 59.57 ± 11.65 | <0.001* |
Gender | |||
Male | 819 (82.1) | 721 (72.6) | <0.001* |
Female | 178 (17.9) | 272 (27.4) | |
Smoking status | |||
Never | 665 (67.6) | 527 (53.1) | <0.001* |
Ever | 319 (32.4) | 466 (46.9) | |
Drinking status | |||
Never | 734 (74.4) | 732 (73.7) | 0.712 |
Ever | 252 (25.6) | 261 (26.3) | |
HBsAg ( |
|||
Negative | 180 (18.9) | ||
Positive | 771 (81.1) | ||
Tumor size ( |
|||
<5 cm | 218 (40.4) | ||
≥5 cm | 322 (59.6) | ||
Tumor number ( |
|||
Single | 475 (88.1) | ||
Multiple | 64 (11.9) | ||
Tumor grade ( |
|||
I-II | 84 (21.8) | ||
III-IV | 301 (78.2) | ||
Serum level of tumor markers | |||
ALT (U/L, in 989 subjects) | 58.29 ± 86.04 | ||
AST (U/L, in 985 subjects) | 62.24 ± 81.06 | ||
AFP | |||
<20 ug/L | 363 (37.2) | ||
≥20 ug/L | 612 (62.8) | ||
(ug/L, in 404 subjects) | 126.87 ± 289.61 (0.6–1210) | ||
HBV-DNA (IU/mL, in 450 subjects) |
|
The genotype distributions of rs6513497 in controls, HCC patients, and HBV-related HCC patients are shown in Table
Association between genotypes/alleles of miR-646 rs6513497 and the risk of HCC.
Genotypes | Controls | HCC patients | HCC patients with HBV | ||||
---|---|---|---|---|---|---|---|
Number (%) | Number (%) | OR (95% CI)a |
|
Number (%) | OR (95% CI)a |
| |
miR-646 rs6513497 |
|
|
|
||||
TT | 795 (80.1) | 825 (82.7) | 1.000 | 639 (82.9) | 1.000 | ||
GT | 186 (18.7) | 166 (16.6) | 0.812 (0.635–1.037) | 0.095 | 126 (16.3) | 0.814 (0.621–1.067) | 0.137 |
GG | 12 (12.2) | 6 (0.6) | 0.468 (0.167–1.308) | 0.147 | 6 (0.8) | 0.631 (0.222–1.793) | 0.387 |
Dominant model |
0.791 (0.622–1.005) | 0.055 | 0.803 (0.616–1.046) | 0.104 | |||
Recessive model |
0.485 (0.174–1.355) | 0.168 | 0.654 (0.230–1.856) | 0.425 | |||
T | 1776 (0.9) | 1816 (0.9) | 1.000 | 1402 (91.0) | 1.000 | ||
G | 210 (0.1) | 178 (0.1) | 0.788 (0.631–0.985) | 0.037* | 138 (9.0) | 0.814 (0.637–1.039) | 0.098 |
aORs and
In males (Table
Comparison of genotype/allele frequencies of miR-646 rs6513497 in male subjects.
Genotypes | Controls | HCC patients | HCC patients with HBV | ||||
---|---|---|---|---|---|---|---|
Number (%) | Number (%) | OR (95% CI)a |
|
Number (%) | OR (95% CI)a |
| |
miR-646 rs6513497 |
|
|
|
||||
TT | 570 (79.1) | 685 (83.6) | 1.000 | 536 (83.4) | 1.000 | ||
GT | 143 (19.8) | 129 (15.8) | 0.692 (0.523–0.915) | 0.010* | 102 (15.9) | 0.719 (0.531–0.973) | 0.033* |
GG | 8 (1.1) | 5 (0.6) | 0.492 (0.151–1.596) | 0.237 | 5 (0.7) | 0.650 (0.198–2.134) | 0.478 |
Dominant model |
0.681 (0.517–0.896) | 0.006* | 0.715 (0.532–0.962) | 0.027* | |||
Recessive model |
0.525 (0.162–1.701) | 0.023* | 0.690 (0.211–2.259) | 0.540 | |||
T | 1283 (89.0) | 1499 (91.5) | 1.000 | 1172 (91.3) | 1.000 | ||
G | 159 (11.0) | 139 (8.5) | 0.695 (0.539–0.897) | 0.005* | 112 (8.7) | 0.739 (0.562–0.972) | 0.030* |
aORs and
Comparison of genotype/allele frequencies of miR-646 rs6513497 in female subjects.
Genotypes | Controls | HCC patients | HCC patients with HBV | ||||
---|---|---|---|---|---|---|---|
Number (%) | Number (%) | OR (95% CI)a |
|
Number (%) | OR (95% CI)a |
| |
miR-646 rs6513497 |
|
|
|
||||
TT | 225 (82.7) | 140 (78.7) | 1.000 | 103 (80.5) | 1.000 | ||
GT | 43 (15.8) | 37 (20.8) | 1.355 (0.817–2.246) | 0.239 | 24 (18.8) | 1.318 (0.727–2.389) | 0.363 |
GG | 4 (1.5) | 1 (0.5) | 0.409 (0.0443–0.799) | 0.431 | 1 (0.7) | 0.597 (0.061–5.833) | 0.657 |
Dominant model |
1.274 (0.777–2.088) | 0.377 | 1.254 (0.702–2.240) | 0.444 | |||
Recessive model |
0.386 (0.042–3.579) | 0.402 | 0.568 (0.058–5.534) | 0.626 | |||
T | 493 (90.6) | 317 (89.0) | 1.000 | 230 (89.8) | 1.000 | ||
G | 51 (9.4) | 39 (11.0) | 1.177 (0.746–1.855) | 0.484 | 26 (10.2) | 1.183 (0.693–2.018) | 0.538 |
aORs and
The association between rs6513497 and HCC or HBV infection was also evaluated by stratifying on other clinical indexes including total bilirubin, HBV-DNA, ALT, AST, and the number, size, and grade of tumor foci. However, no significant heterogeneity was detected between the subgroups, suggesting the independent genetic effect of rs6513497 (Table
Clinicopathologic characteristics and genotype/allele frequencies of miR-646 rs6513497 in HCC patients.
Indexes | Genotype |
|
Allele |
|
|||
---|---|---|---|---|---|---|---|
Tumor size | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.713 | T | G | 0.617 |
<5 cm | 180 (84.1) | 35 (16.9) | 0 (0) | 395 (31.9) | 35 (68.1) | ||
≥5 cm | 261 (82.6) | 53 (16.8) | 2 (0.6) | 575 (34.3) | 57 (65.7) | ||
Tumor focus number | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.195 | T | G | 0.343 |
Single | 396 (83.4) | 78 (16.4) | 1 (0.2) | 870 (91.6) | 80 (8.4) | ||
Multiple | 51 (79.7) | 12 (18.8) | 1 (1.5) | 114 (89.1) | 14 (10.9) | ||
Tumor grade | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.897 | T | G | 0.771 |
I-II | 71 (84.5) | 13 (15.5) | 0 (0) | 155 (92.3) | 13 (7.7) | ||
III-IV | 250 (83.1) | 50 (16.6) | 1 (0.3) | 550 (91.4) | 52 (8.6) | ||
AFP | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.548 | T | G | 0.371 |
<20 ug/L | 294 (81.0) | 67 (18.5) | 2 (0.6) | 655 (90.2) | 71 (9.8) | ||
≥20 ug/L | 511 (83.5) | 97 (15.8) | 4 (0.7) | 1119 (91.4) | 105 (8.6) | ||
Total bilirubin | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.184 | |||
18.79 ± 0.94 | 20.12 ± 3.18 | 21.90 ± 2.86 | |||||
Direct bilirubin | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.347 | |||
8.78 ± 0.70 | 9.91 ± 2.47 | 8.43 ± 1.32 | |||||
Indirect bilirubin | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.125 | |||
9.81 ± 0.24 | 10.22 ± 0.77 | 13.50 ± 1.78 | |||||
ALT | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.643 | |||
57.14 ± 2.79 | 59.92 ± 7.51 | 51.38 ± 8.13 | |||||
AST | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.730 | |||
62.17 ± 2.78 | 63.58 ± 7.04 | 41.50 ± 3.90 | |||||
HBV-DNA | |||||||
miR-646 rs6513497 | TT | GT | GG | 0.526 | |||
|
|
|
Traditional diagnostic methods of HCC have suboptimal efficacy, sensitivity, and specificity [
MiR-646 belongs to miR-15/107 gene group which serves key functions in humans such as cell division, metabolism, stress response, and angiogenesis. Moreover, there is a seed sequence “AGCAGC” near the 5′ end of the mature miR-646 [
Thus, in this study, we investigated the association between rs6513497 and the susceptibility to HCC and HBV-related HCC in a large-scale population. Our findings support the view that rs6513497 was associated with HCC and HBV-related HCC. Furthermore, the protective effect of the variant genotypes of rs6513497 (GT genotype and the G allele) was more evident in male subjects, which decreases their HCC susceptibility. To the best of our knowledge, this is the first study to discover the association of miR-646 SNP with HCC susceptibility in clinical samples, suggesting that rs6513497 could be a useful marker to predict the risk of this disease. Further studies are required to explore the molecular function of this SNP. In addition, previous studies found that the SNPs of pri-miR-218 [
Using TargetScan [
In conclusion, the results of this study suggest that the miR-646 SNP rs6513497 is associated with HCC, and the G allele may be a genetic protective factor which decreases the susceptibility of HCC, especially in male subjects. To better understand the relationship between rs6513497 and cancer risk, more functional studies of miR-646 and this SNP are proposed.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Rui Wang, Jun Zhang, and Weiru Jiang contributed equally to this work.
This work is supported by Grants from Ministry of Science and Technology of China (no. 2012CB910104) and National Natural Science Foundation of China (nos. 81300327 and 81201895). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the paper.