Molecular Biology Reports

, Volume 39, Issue 6, pp 7019–7023

Association of the microRNA-499 variants with susceptibility to hepatocellular carcinoma in a Chinese population

Authors

  • Yu Xiang
    • Department of Laboratory MedicineThe First Affiliated Hospital of Chongqing Medical University
  • Song Fan
    • Department of Laboratory MedicineThe First Affiliated Hospital of Chongqing Medical University
  • Ju Cao
    • Department of Laboratory MedicineThe First Affiliated Hospital of Chongqing Medical University
  • Shifeng Huang
    • Department of Laboratory MedicineThe First Affiliated Hospital of Chongqing Medical University
    • Department of Laboratory MedicineThe First Affiliated Hospital of Chongqing Medical University
Article

DOI: 10.1007/s11033-012-1532-0

Cite this article as:
Xiang, Y., Fan, S., Cao, J. et al. Mol Biol Rep (2012) 39: 7019. doi:10.1007/s11033-012-1532-0

Abstract

microRNAs (miRNAs) are a new class of small non-coding RNAs that function as tumor suppressors or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNA may contribute to cancer development. We hypothesized that genetic variations of the miRNA could be associated with the risk of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). A total of 100 patients with HCC, 100 cases of chronic hepatitis B and 100 health adults were enrolled in this present study. Two common polymorphisms in pre-miRNAs: Homo sapiens miRNA-146a (hsa-mir-146a) (rs291016, guanine to cytosine [G-C]) and hsa-mir-499 (rs3746444; adenine to guanine [C-T]) were genotyped by PCR-Restriction Fragment Length Polymorphism and confirmed by bidirectional DNA sequencing. Significant differences were found in frequency and distribution of the genotypes of miRNA-499 between the HCC and the control group. Compared with miRNA-499 T/T, the odds ratio (OR) of patients with miRNA-499 C/C for developing HCC was 3.630 (95% CI: 1.545–8.532), and OR for developing HBV-related HCC was 3.133 (95% CI: 1.248–7.861). There was no significant association between miRNA-146a polymorphism and the risk of HCC in all subjects. Our results suggested that hsa-mir-499 polymorphism was associated with susceptibility to HBV-related HCC in Chinese population. Further characterization of miRNA SNPs may open new avenue for the study of cancer and therapeutic interventions.

Keywords

Hepatitis B virusHepatocellular carcinomaSingle nucleotide polymorphismsmicroRNAsmiRNA-146amiRNA-499

Introduction

Hepatitis B virus (HBV) infection is an important worldwide public health problem. Exposure to HBV can cause variable clinical conditions including spontaneous recovery from acute hepatitis, asymptomatic carrier, chronic hepatitis, liver cirrhosis or hepatocellular carcinoma (HCC) [1]. HCC is the fourth most common cause of death from cancer and China alone accounts for 53% of all liver cancer death worldwide [2]. It has been shown that both genetic and environmental factors are involved in the etiology and prognosis of HCC [3].

microRNAs (miRNAs) are a group of small non-coding RNA molecules which have been identified in many organisms and could regulate the expression of genes in a variety of eukaryotic systems [4, 5]. miRNAs play crucial roles in many physiological and pathological conditions, such as development [6], cellular differentiation [7], proliferation [8], cell death [911] and metabolism [12]. Numerous studies have demonstrated that alteration of miRNAs plays a critical role in cancer development [13, 14] through regulating the expressions of proto-oncogenes or tumor suppressor genes [1315].

SNP is the most common type of genetic variation in human genome. It has been well demonstrated that SNPs in protein-coding genes can affect the functions of proteins and in turn influence the individual susceptibility to cancers [16]. A G>C polymorphism has been identified in the miR-146a gene, and this polymorphism is located in the stem region opposite to the mature miR-146a sequence which results in a change from G:U pair to C:U mismatch in the stem structure of miR-146a precursor. A C>T polymorphism has been identified in the miR-499 gene, and this polymorphism is located in the stem region opposite to the mature miR-499 sequence which results in a change from A:U pair to G:U mismatch in the stem structure of miR-499 precursor. Whether sequence variations of miRNA genes are also involved in tumorgenesis is still unknown. Therefore, we hypothesized that SNPs in these miRNAs may contribute to susceptibility to HCC. To test this hypothesis, we genotyped and analyzed the associations of polymorphisms in miR-499 and miR-146a with the risk of HCC in a hospital-based, case–control study of 100 patients with HCC and a control group of 100 cancer-free individuals in a Chinese population.

Materials and methods

Subjects

A total of 300 subjects were periodically enrolled between December 2009 and February 2011 at the First Affiliated Hospital of Chongqing Medical University, including 100 patients with HCC, 100 cases of chronic hepatitis B (CHB) and 100 health adults. The HCC patients were divided into two groups: those without HBV (n = 27) and those with HBV (n = 73). CHB patients were positive for HBsAg. All patients did not have any other types of liver diseases, such as chronic hepatitis C, alcoholic liver diseases, autoimmune liver diseases, or metabolic liver diseases. The diagnosis of HCC was histopathologically confirmed. Data on all subjects were obtained from medical records, pathology reports, and personal interviews with the subjects. The data collected include age, gender, alpha fetoprotein (AFP) level, aspartate aminotransferase (AST) level, alanine aminotransferase (ALT) level and HBV-DNA level.

Genotyping of sequence variants

Genomic DNA was isolated from whole blood using a commercial purification kit (Qiagen, Hilden, Germany), and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to determine the sequence variants of miRNA-146a and miRNA-499, followed by genetic confirmation through bi-directional DNA sequencing. All primer sequences and PCR conditions were listed as in Table 1.
Table 1

Primer sequences and amplification conditions of the genes

Genes (variants)

Primer sequence (5′–3′)

Initial denaturation

Denaturation

Annealing

Extension

Cycling number

miRNA-146a (G-C)

F:5′-CATGGGTTGTGTCAGTGTCAGAGCT-3′

94°C

5 min

94°C

1 min

54°C

45 s

72°C

1 min

35

R:5′-TGCCTTCTGTCTCCAGTCTTCCAA-3′

miRNA-499 (T-C)

F:5′-CAAAGTCTTCACTTCCCTGCCA-3′

R:5′-GATGTTTAACTCCTCTC CACGTGATC-3′

miRNA-146a amplification products of 147 bp (the GG genotype) were digested by restriction enzymes SacI (Takara Company, Japan) into fragments of 122 and 25 bp for CC and into fragments of 147, 122, and 25 bp for GC. miRNA-499 PCR amplicons of 146 bp (the CC genotype) were digested by BclI (Takara Company, Japan) into fragments of 120 and 26 bp for TT and into fragments of 146, 120 and 26 bp for CT. The digestion products were separated by 3% agarose gel electrophoresis. After Goldview staining, DNA bands were observed and analyzed by gel imager.

Statistical analysis

All genotype and allele frequencies were calculated by frequency counting method and then verified by Hardy–Weinberg law. Comparisons were made by χ2 test and independent samples T test. P < 0.05 was considered statistically significant. The risk of each genotype for developing HCC was expressed with odds ratio (OR) and 95% confidence interval (CI). OR value was calculated by logistic regression analysis. All statistical tests were bilateral probability test.

Results

General clinical characteristics

The clinical details and the results of the biochemical analyses from the Control, CHB and HCC subjects at the time of the study were shown as in Table 2. No significant difference was found in age between CHB, HCC and Control group. HCC group had a higher proportion of male subjects. In addition, HCC group and other two groups (Control and CHB group) had statistically different laboratory results for sex, ALT and AST (P < 0.05).
Table 2

Clinical characteristics of CHB, HCC patients and controls

 

Control

n = 100

CHB

n = 100

HCC

n = 100

P

Age

45.12 ± 15.82

47.02 ± 14.48

48.55 ± 9.29

>0.05

Sex

 Male

50 (50%)

39 (39%)

82 (82%)

<0.05

 Female

50 (50%)

61 (61%)

18 (18%)

 

ALT (IU/l)

9.75 ± 4.71

31.64 ± 43.21

91.83 ± 138.74

<0.05

AST (IU/l)

10.47 ± 5.14

35.82 ± 52.42

116.65 ± 177.32

<0.05

The HCC patients were further divided into two groups: those with HBV infection (HBV+ HCC n = 73) and those without HBV infection (HBV HCC n = 29). While no significant difference was found in age, sex, ALT and AST between the HBV+ HCC and the HBV HCC group, significant differences were found for both AFP and HBV-DNA between the two groups (Table 3).
Table 3

Clinical characteristics of HBV+ HCC and HBV HCC patients

 

HBV+ HCC

n = 73

HBV HCC

n = 27

P

Age

48.99 ± 8.84

47.37 ± 10.52

>0.05

Sex

 Male

63 (86%)

19 (70%)

>0.05

 Female

10 (14%)

8 (30%)

 

ALT (IU/l)

96.44 ± 125.52

79.37 ± 171.63

>0.05

AST (IU/l)

130.00 ± 166.38

80.56 ± 167.60

>0.05

AFP (ng/ml)

17015.17 ± 62954.54

1004.35 ± 3396.74

<0.05

HBV-DNA (IU/ml)

4.80 × 105 ± 1.68 × 106

1.40 × 103 ± 1.90 × 103

<0.05

Frequency and distribution of the genotypes and alleles of the miRNA-146a and miRNA-499 gene in HCC, CHB and control group

χ2 test demonstrated that all the six genotypes and allele frequencies were consistent with the Hardy–Weinberg law. Table 4 shows the differential frequencies and distributions of genotypes of miRNA-146a and miRNA-499 in the three study groups. While no significant difference was found for the distribution of miRNA-146a variants among CHB, HCC patients and healthy controls, the frequency of miRNA-499 C allele was found to be significantly higher in HCC group and HBV+-HCC patients than that in the control group (χ2 = 9.575, P < 0.05; χ2 = 6.884, P < 0.05; respectively).
Table 4

Genotype and allele frequencies of miRNA-146a and miRNA-499 in the case and control groups

Genotype

Control

n = 100

CHB

n = 100

HCC

n = 100

HBV+-HCC

n = 73

miRNA-146a

 

χ2 = 3.040, P > 0.05

χ2 = 1.171, P > 0.05

χ2 = 0.496, P > 0.05

 G/G

21 (0.21)

24 (0.24)

27 (0.27)

18 (0.25)

 G/C

46 (0.46)

54 (0.54)

45 (0.45)

34 (0.46)

 C/C

33 (0.33)

22 (0.22)

28 (0.28)

21 (0.29)

Alleles

 G

88 (0.44)

102 (0.51)

170 (0.85)

70 (0.48)

 C

112 (0.56)

98 (0.49)

30 (0.15)

76 (0.52)

miRNA-499

 

χ2 = 0.443, P > 0.05

χ2 = 9.575, P < 0.05

χ2 = 6.884, P < 0.05

 T/T

54 (0.54)

52 (0.52)

36 (0.36)

27 (0.37)

 T/C

36 (0.36)

35 (0.35)

40 (0.40)

30 (0.41)

 C/C

10 (0.10)

13 (0.13)

24 (0.24)

16 (0.22)

Alleles

 T

144 (0.72)

139 (0.70)

112 (0.56)

84 (0.58)

 C

56 (0.28)

61 (0.30)

88 (0.44)

62 (0.42)

miRNA-499 C/C was associated with susceptibility to HCC and HBV-related HCC

Table 5 shows the differential risks of each variant of miRNA-499 and miRNA-146a in the three study groups. While no significant difference was found in ORs of the miRNA-146a variants among HCC, CHB patients and controls, miRNA-499 C/C were shown to be associated with a higher susceptibility to HCC. Moreover, OR for HCC patients with miRNA-499 C/C in developing HCC was 3.63 (95% CI: 1.545–8.532) when compared with miRNA-499 T/T. On the other hand, OR for HBV-related HCC patients with miRNA-499 C/C in developing HBV-related HCC was 3.13 (95% CI: 1.248–7.861) when compared to miRNA-499 T/T.
Table 5

Risks of miRNA-146a and miRNA-499 polymorphisms for the development of HCC

Genotype

Risk for HCC

OR (95%CI)

Risk for HBV+-HCC

OR (95%CI)

Risk for CHB

OR (95% CI)

mir-146a

 C/C

1.00

1.00

1.00

 G/G

1.522 (0.698–3.319)

1.241 (0.527–2.920)

1.718 (0.771–3.824)

 G/C

1.086 (0.557–2.116)

1.086 (0.529–2.229)

1.733 (0.886–3.392)

mir-499

 T/T

1.00

1.00

1.00

 C/C

3.630 (1.545–8.532)#

3.133 (1.248–7.861)$

1.288 (0.514–3.227)

 T/C

1.693 (0.911–3.140)

1.674 (0.856–3.272)

1.043 (0.567–1.917)

#P < 0.001; $ P < 0.001

Discussion

In China, HCC ranks the second among all malignancies and HBV infection is highly endemic [17]. The worldwide incidence of HCC is much higher in male compared with female individuals. Our results suggested that HCC group had a higher proportion of male subjects. The levels of AFP and HBV-DNA were significantly higher in HBV+ HCC group than those in HBV HCC group. HBV is the major cause of HCC. However, the molecular mechanism of virus-induced carcinogenesis is still poorly understood.

The non-coding small RNAs may lead novel insights into the biologic mechanism of HCC. Although the association between SNPs in protein-coding genes and the risk of cancers has been investigated extensively, few studies concerning the association of SNPs in miRNA genes with the risk of cancers have been reported. Here, we analyzed the influence of miR-146a and miR-499 polymorphisms on individual susceptibility to HCC. In the present study, while no significant difference was found in ORs of the miRNA-146a variants among HCC, CHB patients and controls, miRNA-499 C/C was shown to be associated with a higher susceptibility to HCC. Moreover, we found that HBV+ individuals with CC genotype of miR-499 gene were more susceptible for HCC compared with those carrying TT genotype.

miRNA profiling studies have revealed that many miRNAs are up- or down-regulated in different types of human cancers and that most of them are down-regulated [18]. Since each miRNA has numerous targets, inherited minor variations in miRNA expression could have important consequences on the expression of various protein coding oncogenes and tumor suppressor genes involved in malignant transformation. SNP in miRNA-146a contributes to the genetic predisposition to papillary thyroid carcinoma [19]. A functional polymorphism in the miR-146a gene was associated with the risk for HCC in male patients [20]. The reason behind such discrepancy may be due to the different population in different areas and case groups we had chosen.

Early detection of high risky individuals in the population is one of the best strategies for preventing cancer. SNPs or mutations in miRNA sequence may alter miRNA expression and/or maturation, and thus contribute to the susceptibility to cancers. Indeed, the rs3746444 SNP in miRNA-499 and rs291016 SNP in miRNA-146a have been reported to be associated with the susceptibility of squamous cell carcinoma of the head and neck [21]. In our study, the results revealed that individuals with miRNA-499 CC genotype were about threefold more susceptible to HCC (OR 3.63, 95% CI 1.545–8.532) compared with those with TT genotype. Besides, OR of HBV-related HCC patients with miRNA-499 C/C for developing HBV-related HCC was 3.13 (95% CI: 1.248–7.861) when compared with miRNA-499 T/T.

In conclusion, our results suggested that miRNA-499 polymorphism was associated with the susceptibility to HBV-related HCC in a Chinese population. This is the first report of the association between miRNA-499 polymorphism and HBV-related HCC in Chinese population, and further prospective investigations with a large number of cases would allow us to evaluate miRNA-499 polymorphism in a variety of clinical settings to help us better understand its role in HCC.

Acknowledgments

This work was supported in part by the National Science Foundation of China (Grant No. 81071621 and 30973378), the Natural Science Foundation of Chongqing, China (Grant No. CSTC, 2010BB5390), the Science Foundation of Chongqing Municipal Bureau of Health (Grant No. 2010-2-090) and the Medical Science Foundation of the First Affiliated Hospital of Chongqing Medical University (Grant No. YXJJ 2009-12).

Copyright information

© Springer Science+Business Media B.V. 2012