Association Between pri-miR-218 Polymorphism and Risk of Hepatocellular Carcinoma in a Han Chinese Population

Abstract

MicroRNAs are noncoding RNA molecules of 18–25 nucleotides that regulate gene expression at the post-transcriptional level. The aim of this study was to investigate whether pri-miR-218 rs11134527 A/G polymorphism influences the risk of hepatocellular carcinoma (HCC) or not. pri-miR-218 rs11134527 A/G was genotyped in 302 HCC patients and 513 control subjects using the polymerase chain reaction–restriction fragment length polymorphism assay. The AG genotype of pri-miR-218 rs11134527 A/G was associated with family history (p=0.018, odds ratio [OR]=2.96, 95% confidence interval [CI]: 1.16–7.56) and elevated serum α-fetoprotein (serum alpha-fetoprotein [AFP]) levels (≥20 ng/mL; p=0.009, OR=1.92, 95% CI: 1.17–3.14) in HCC patients. These findings suggested that the AG genotype of pri-miR-218 rs11134527 might relate to genetic predisposition and be involved in regulating the expression of AFP in Chinese HCC patients.

Introduction

P rimary liver cancer or hepatocellular carcinoma (HCC) is the fifth most common solid cancer worldwide, the third most frequent cause of cancer death in men, and the sixth in women. Approximately 650,000 people die from HCC each year, and > 75% of these cases occur in the Asia-Pacific region (Yuen et al., 2009). China alone accounts for 55% cases of HCC worldwide (Parkin, 2001). The tumorigenesis of HCC was related to comorbidities such as chronic hepatitis B, chronic hepatitis C, chronic alcohol abuse, cigarette smoking, family history of HCC, diabetes mellitus, environmental factors, and unknown genetic factors (Donato et al., 1999; Wang et al., 2003; Marrero et al., 2005; Amarapurkar et al., 2008; Yuen et al., 2009). In China, hepatitis-B virus (HBV) infection is highly endemic; therefore, HCC ranks the second among all malignancies (Srivatanakul et al., 2004).

The methods of diagnosing HCC include magnetic resonance imaging and/or computed tomography scan of the abdomen, serum alpha-fetoprotein (AFP) measurement, and liver biopsy. Serum AFP has been widely used for the diagnosis and for evaluating the prognosis of HCC (Johnson, 2001; Farinati et al., 2006). However, the sensitivity and specificity of AFP in HCC diagnosis depends on the cutoff level used. Farinati et al. (2006) divided the patients into three groups according to their AFP levels: normal (<20 ng/mL), elevated (21–400 ng/mL), and diagnostic (>400 ng/mL). They found that AFP levels were within normal range in 46% of the cases, between normal range and diagnostic levels (21–400 ng/mL) in 36%, and above 400 ng/mL in 18%. They confirmed that AFP was a marker of low sensitivity (54%) in the HCC diagnosis (Farinati et al., 2006). Previous studies reported that AFP might help HCC escape from immunological control by inhibiting of apoptosis and induce functional impairment in the cancer cells; hence, AFP might participate in the promotion of tumor development (Behboudi et al., 2004; Li et al., 2005).

MicroRNAs (miRNAs) consist of noncoding RNA molecules of 18–25 nucleotides emerging after multiple steps of maturation and are important regulatory molecules in animals and plants (Ambros, 2004). miRNAs regulate gene expression at the post-transcriptional level by binding to partially complementary sequences in the 3'-untranslated region (3′-UTR) of target mRNAs (Gaul et al., 2007). Recent work has demonstrated that miRNAs are involved in various biological and pathological processes, including development timing, cellular differentiation, proliferation, apoptosis, insulin secretion, cholesterol biosynthesis, and tumorigenesis (Cai et al., 2009). Previous data showed that miR-218 was expressed throughout the embryonic development of the liver and pancreas by binding to target site in the 3′-UTR of Onecut transcription factor 2 (OC-2) and suggested that miR-218 might have an critical role in the process of liver and pancreas development (Simion et al., 2010). Moreover, several studies had focused on the level of miR-218 RNA expression changes, and they found that miR-218 might act as a tumor suppressor gene (Zanette et al., 2007; Gao et al., 2009; Liu et al., 2009; Davidson et al., 2010; Song et al., 2010; Leite et al., 2011).

Single-nucleotide polymorphism (SNP) is the most abundant form of DNA variation in the human genome and contributes to human phenotypic differences. Polymorphisms in miRNA genes, including pri-miRNAs (primary miRNAs), premiRNAs (precursor miRNAs), and mature miRNAs, potentially alter various biological processes by influencing the processing and/or target selection of miRNAs (Iwai and Naraba 2005; Mishra and Bertino 2009). To date, high-throughput miRNA microarray and quantitative real-time polymerase chain reaction (PCR) have been adopted to detect differential expression of miRNAs and the expression level of miRNAs. To our knowledge, no investigation of the association between the pri-miR-218 polymorphism and the risk of HCC has been undertaken. In the present study, we investigated the influence of pri-miR-218 rs11134527 A/G polymorphism on the risk of HCC in a Han Chinese population.

Materials and Methods

Subjects and clinical characteristics

This study enrolled 302 unrelated HCC patients hospitalized in the West China Hospital of Sichuan University between June 2008 and October 2010. All patients involved in this study were pathologically confirmed. The blood sample was taken before their initial treatment. Clinical characteristics, including gender, age, family history of HCC, serum AFP levels, and HBV serological markers (HBs Ag [hepatitis-B surface antigen], HBs Ab [hepatitis-B surface antibody], HBe Ag [hepatitis-B e antigen], anti-HBe Ab [antibody to hepatitis-B e antigen] and anti-HBc [antibody to hepatitis-B core antigen]), were collected. The control group was recruited from a routine health survey with a total number of 513 unrelated individuals, and inclusive criteria for controls were no evidence of any personal or family history of cancer or other serious diseases. All subjects were from the Han population living in Sichuan Province of southwestern China. This study was approved by the ethics committee of West China Hospital, Sichuan University, and all subjects gave written informed consent for participation.

Genotyping

Genomic DNA was extracted from peripheral blood with an extraction kit (Bioteke Corporation, Beijing, China) according to the manufacturer's instructions. The pri-miR-218 rs11134527 polymorphism was identified by PCR–restriction fragment length polymorphism analysis. The amplification was performed using primers previously published (Zhou et al., 2010). The PCR was performed in a total volume of 25 μL, including 2.5 μL of 10X PCR buffer, 1.5 mM MgCl₂, 0.15 mM dNTPs, 0.5 μM each primer, 100 ng of genomic DNA, and 1 U of Taq DNA polymerase. The reactions were incubated at 94°C for 4 min, followed by 35 cycles at 94°C for 30 s, 66.5°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. PCR products were digested with BclI (New England BioLabs, Beverly, MA) for 4 h. The length of digestion products was previously described (Zhou et al., 2010). To confirm the accuracy of the method we used, about 10% of the samples were randomly selected for repeated experiments and the results were 100% concordant.

Statistical analysis

All statistical analyses were performed by SPSS version 11.5 (SPSS, Chicago, IL). The χ ² test was applied to evaluate Hardy–Weinberg equilibrium in all subjects, genotype and allele frequencies, and clinical characteristics between groups. A p-value of <0.05 was considered statistically significant.

Results

Subjects and clinical characteristics

A total of 302 HCC patients and 513 control cases were involved in this study. No significant differences between the two groups in terms of gender and age distribution suggested that matching of subjects based on these variables was adequate. The male-to-female ratio of HCC patients is 6:1, with a median age of 57 (male) and 51 (female) years, respectively. Among the patients, 227 (75.2%) were HBs Ag positive, 27 (8.9%) had family history of HCC, 95 (31.5%) with normal serum AFP levels (<20 ng/mL), 75 (24.8%) with elevated serum AFP levels (21–400 ng/mL), and 132 (43.7%) with diagnostic serum AFP levels (≥400 ng/mL). The general characteristics of the population are shown in Table 1.

Table 1.

Characteristics of the Hepatocellular Carcinoma Patients and Controls

	HCC patients		Controls
Characteristics	n=302	%	n=513	%	OR (95% CI)	χ² -value	p-Value
Gender (n)
Male	258	85.4	415	83.8	1.00 (Ref.)
Female	44	14.6	98	14.2	0.72 (0.5–1.06)	2.71	0.10
Age (years)
Mean±SD	51.8±12.7		48.2±15.8
< 40	48	15.9	105	20.5	1.00 (Ref.)
≥40	254	84.1	408	79.5	1.36 (0.94–1.98)	2.61	0.11
Family history of HCC
Yes	27	8.9
No	275	91.1
HBV serological markers
HBs Ag(+)	227	75.2
HBs Ag(−)	75	24.8
Serum AFP
< 20 (ng/mL)	95	31.5
21–400 (ng/mL)	75	24.8
≥400 (ng/mL)	132	43.7

OR, odds ratio; CI, confidence interval; SD, standard deviation; HBs Ag, hepatitis-B surface antigen; HCC, hepatocellular carcinoma; HBV, hepatitis-B virus; AFP, alpha-fetoprotein.

pri-miR-218 rs11134527 polymorphism and risk of HCC

The genotype distribution in the control group was in Hardy–Weinberg equilibrium (p=0.07). The genotype and allele frequencies of the pri-miR-218 rs11134527A/G did not differ significantly between the HCC patients and the control group (Table 2).

Table 2.

pri-mir-218 rs11134527 A/G Genotypes of the Hepatocellular Carcinoma Patients and Controls

Polymorphism	Genotype/allele	HCC patients	Controls	OR (95% CI)	χ² Value	p-Value
pri-mi218 rs11134527A/G		n=302 (%)	n=513 (%)
	AA	88 (29.1)	165 (32.2)	1.00 (Ref.)
	AG	170 (56.2)	269 (52.4)	1.19 (0.86–1.64)	1.07	0.30
Genotypes	GG	44 (14.6)	79 (15.4)	1.04 (0.67–1.50)	0.04	1.63
	A	346 (57.3)	599 (58.4)	1.00 (Ref.)
Alleles	G	258 (42.7)	427 (41.6)	1.04 (0.85–1.28)	0.19	0.67

pri-miR-218 rs11134527 polymorphism and clinical characteristics of HCC

After stratified analyses according to clinical characteristics, we observed that the AG genotype of pri-miR-218 rs11134527 was associated with family history (p=0.018, odds ratio [OR]=2.96, 95% confidence interval [CI]: 1.16–7.56) and elevated serum AFP levels (≥20 ng/mL; p=0.009, OR=1.92, 95% CI: 1.17–3.14) in HCC patients. However, we found no significant association between the pri-miR-218 rs11134527 polymorphism and other factors such as gender, age, and HBV infection status (Table 3).

Table 3.

Stratification Analyses of the pri-mir-218 rs11134527 A/G Polymorphism with Clinical Characteristics in Hepatocellular Carcinoma Patients

	Genotype frequency
Characteristics	n (%)	n (%)	OR (95% CI)	χ² Value	p-Value
Gender	Male	Female
AA+GG	112 (43.4%)	20 (45.5%)
AG	146 (56.6%)	24 (54.5%)	1.08 (0.57–2.07)	0.06	0.80
A allele	294 (57.0%)	52 (59.0%)	1.00 (Ref.)
G allele	222 (43.0%)	36 (41.0%)	1.09 (0.69–1.73)	0.14	0.71
Age	<40	≥40
AA+GG	20 (41.7%)	112 (44.0%)	1.00 (Ref.)
AG	28 (58.3%)	142 (56.0%)	1.10 (0.59–2.06)	0.10	0.76
A allele	56 (58.3%)	290 (57.1%)	1.00 (Ref.)
G allele	40 (41.7%)	218 (42.9%)	0.95 (0.61–1.48)	0.51	0.82
Family history of HCC	Yes	No
AA+GG	6 (22.2%)	126 (45.8%)	1.00 (Ref.)
AG	21 (77.8%)	149 (54.2%)	2.96 (1.16–7.56)	5.56	0.018^*
A allele	27 (50%)	319 (58%)	1.00 (Ref.)
G allele	27 (50%)	231 (42%)	1.38 (0.79–2.42)	1.29	0.26
HBV	HBs Ag (+)	HBs Ag (−)
AA+GG	100 (44.0%)	32 (42.7%)	1.00 (Ref.)
AG	127 (56.0%)	43 (57.3%)	0.95 (0.56–1.60)	0.04	0.83
A allele	251 (55.3%)	95 (63.3%)	1.00 (Ref.)
G allele	203 (44.7%)	55 (36.7%)	1.40 (0.96–2.04)	2.98	0.084
Serum AFP	≥20	<20
AA+GG	80 (39.6%)	52 (54.8%)	1.00 (Ref.)
AG	127 (61.4%)	43 (45.2%)	1.92 (1.17–3.14)	6.85	0.009^*
A allele	229 (55.3%)	117 (67.6%)	1.00 (Ref.)
G allele	185 (44.7%)	73 (38.4%)	1.30 (0.91–1.84)	2.09	0.15

p<0.05 for χ² test.

Discussion

In this study, we identified the association between a pri-miRNA SNP and the risk of HCC, as well as the HCC patients' characteristics. There was no significant association in genotype and allele frequencies of the pri-miR-218 rs11134527A/G polymorphism between the HCC patients and the control group; however, we found that the AG genotype was associated with family history and elevated AFP levels (≥20 ng/mL) in HCC patients.

QUANTO software was used to estimate the statistical power of association between the pri-miR-218 rs11134527 A/G polymorphism and risk of HCC. Our study had 89% power to detect the effect with a relative risk of 1.6 under a dominant genetic model.

miR-218 is found in the brain and regulates the expression of OC-2, which is an important regulator of liver and pancreas development (Sempere et al., 2004; Simion et al., 2010). Previous studies have found reduced expression of the miR-218 in gastric cancer, glioma cells, lung squamous cell carcinoma, prostate carcinoma, and medulloblastomas, but increased expression in leukemia (Zanette et al., 2007; Gao et al., 2009; Liu et al., 2009; Davidson et al., 2010; Song et al., 2010; Leite et al., 2011). miR-218 expression was significantly reduced in gastric cancer tissues, Helicobacter pylori-infected gastric mucosa, and H. pylori-infected AGS cells. Interestingly, overexpression of miR-218 inhibited cell proliferation and increased apoptosis in vitro (Gao et al., 2009). Song and colleagues (2010) found that upregulation of miR-218 drastically reduced the migration and invasion of glioma cells, whereas suppression of miR-218 enhanced the invasive ability of glioma cells. Further, miR-218 reduced the levels of Laminin 5β3 (LAMB3) mRNA as well as the protein. LAMB3 increased cell migration and tumorigenicity in severe combined immune deficiency mice and promoted tumorigenesis in human keratinocytes (Khan et al., 2008). All the aforementioned findings suggested that miR-218 might take a key role in tumorigenesis, invasion, and metastasis; accordingly, it might act as a tumor suppressor.

Polymorphisms present in pri-, pre-, and mature-miRNA can potentially influence expression of hundreds of genes and pathways and broadly affect miRNA function (Duan et al., 2007). A series of studies about polymorphism in pri-miRNAs have been published (Harnprasopwat et al., 2010; Zhou et al., 2010). Zhou et al. (2010) genotyped pri-miR-218 rs11134527 and found it might contribute to cervical cancer carcinogenesis in Chinese women. In this study, significant association was found between the AG genotype of pri-miR-218 rs11134527 and family history in HCC patients, suggesting that the AG genotype of pri-miR-218 rs11134527 was related with genetic predisposition in HCC patients. Further, elevated serum AFP level (≥20 ng/mL) associated with the AG genotype of pri-miR-218 rs11134527 was also found. AFP levels elevate during embryogenesis, but declined rapidly after birth (Gillespie and Uversky 2000). However, the AFP gene is reactivated in patients with HCC (Farinati et al., 2006), embryonic carcinomas (Chiriva-Internati et al., 2007), gastric cancer (Chen et al., 2003), and lung cancer (Hiroshima et al., 2002), and high levels of AFP have been found in serum and tumor tissues. Regulation of AFP gene expression is a complex process and is mediated by a number of transcriptional activators and repressors that bind to a complex AFP promoter and enhancer (Chen et al., 2007). SNPs located at the binding sites for the transcription factors might be related to the production of AFP and the growth of cancer. We found that the AG genotype of pri-miR-218 rs11134527 was associated with elevated serum AFP level (≥20 ng/mL), suggesting that the AG genotype of pri-miR-218 rs11134527 might be involved in regulating the expression of AFP.

In summary, we found that the AG genotype of pri-miR-218 rs11134527 was associated with family history and elevated AFP levels (≥20 ng/mL) in Han Chinese HCC patients. Nevertheless, further studies should be carried out to confirm these findings with larger sample size, especially in different populations. Additionally, more pri-miRNAs should be genotyped to improve our knowledge on miRNA biogenesis and the potential contribution of these SNPs to HCC etiology and progression.

Footnotes

Acknowledgment

The authors thank all the people who participated in this study.

Author Contributions

L.-S. Zhang, W.-B. Liang, and L.-B. Gao conceived the study, participated in its design, carried out most of the experiments, and drafted the article. L. Zhang participated in the study's design and helped to draft the article. H.-Y. Li and Y.-G. Wei collected the blood samples. P.-Y. Chen, L.-J. Li, and J.-G. Han collected the clinical characteristics and data of samples and performed DNA extraction. Y. Liu and T.-Y. Chen participated in genotyping and statistical analysis. All authors have read and approved the final article.

Disclosure Statement

No competing financial interests exist.

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