Association of miRNA-122-binding site polymorphism at the interleukin-1 α gene and its interaction with hepatitis B virus mutations with hepatocellular carcinoma risk

Abstract

This study was designed to investigate the contribution of miRNA-122-binding site polymorphism at the IL-1A gene and its multiplicative interactions with hepatitis B virus (HBV) mutations in the risk of hepatocellular carcinoma (HCC). A total of 1021 healthy controls, 302 HBV surface antigen (HBsAg) seroclearance subjects, and 2011 HBsAg-positive subjects (including 1021 HCC patients) were enrolled in this study. Quantitative PCR was used to genotype rs3783553. HBV mutations were determined by direct sequencing. Multivariate logistic regression analyses were performed to test the associations of rs3783553, mutations, and their interactions with the risk of HCC. No significant association was found between rs3783553 and the risk of HCC among healthy controls, HBsAg seroclearance subjects, HBsAg-positive subjects without HCC, and all controls. Additionally, rs3783553 was not significantly associated with chronic HBV infection, liver cirrhosis, HBV e antigen seroconversion, abnormal alanine aminotransferase, and high viral load (>10⁴ copies/ml). However, the TTCA insertion allele of rs3783553 was significantly associated with an increased frequency of HBV C7A mutation compared with homozygous TTCA deletion carriers [(del/ins+ ins/ins) vs. del/del, adjusted odds ratio (OR)=1.48, 95% confidence interval (CI)=1.09-2.02, P=0.013]. Multiplicative interaction of rs3783553 with HBV preS deletion significantly reduced the risk of HCC in males, with an adjusted OR of 0.64 (95% CI=0.42-0.98; P=0.041) after age and HBV genotype were adjusted. Although rs3783553 did not significantly affect genetic susceptibility to HBV-related HCC, its variant allele may predispose the host to selecting HBV C7A mutation during evolution and significantly reduce the risk of HCC caused by HBV preS deletion. This study provides an insight into the complex host-virus interaction in HBV-induced hepatocarcinogenesis and is helpful in determining HBsAg-positive subjects who are likely to develop HCC.

Keywords

miRNA-122-binding site / IL-1A / rs3783553 / hepatitis B virus (HBV) mutations / hepatocellular carcinoma (HCC) / interaction

Introduction

Chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is the leading cause of hepatocellular carcinoma (HCC) worldwide [1,2]. The mainland of China, a region with 94 million HBV surface antigen (HBsAg) carriers, alone accounts for half of the global HCC cases [3,4]. The cumulative lifetime (age: 30 to 75 years) incidence rates of HCC for males and females who were only positive for HBsAg were 27.38% and 7.99%, whereas those without HBV and HCV infections were 1.55% and 1.03%, respectively [5]. In East Asia, where HBV genotypes B and C are endemic, active and persistent inflammation in the liver, high levels of circulating HBV DNA (≥10⁴ vs.<10⁴ copies/ml), HBV genotype C (vs. genotype B), and viral mutations in the enhancer II/basal core promoter/precore (EnhII/BCP/PC) and the preS regions of HBV are significantly associated with increased risks of HCC [5-12].

The chronic inflammation caused by the interaction of HBV infection and subverted host immunity creates a microenvironment that facilitates hepatocarcinogenesis and HCC late recurrence [13-15]. Antiviral treatments with either class I interferon or nucleot(s)ide analogs not only prevent the occurrence of HCC but also improve postoperative survival [16-19]. HBV replication might promote active inflammation by dysregulating proinflammatory cytokines or chemokines. Of those, interleukin-1 (IL-1) family members have been associated with chronic inflammation, carcinogenesis, and metastasis [20]. MicroRNAs (miRNAs), a group of noncoding RNA molecules that are 18 to 25 nucleotides long, have post-transcriptional regulatory functions through binding to the 3′ untranslated region (UTR), coding region, or 5′UTR of the target mRNAs; miRNAs are also involved in HBV-induced inflammatory diseases and hepatocarcinogenesis [21,22]. miRNAs regulate HCC-promoting inflammation possibly by regulating proinflammatory signaling pathways, such as nuclear factor-κB [23]. Functional polymorphisms within miRNA binding sites may influence the susceptibility of an individual to cancer by altering the strength of miRNA binding and regulating target genes [24].

miRNA-122, a liver-specific miRNA, is related to viral immune escape and anti-viral defense [10,25]. rs3783553, a miRNA-122 binding site polymorphism at the IL-1A gene, has been reported to be associated with risk of HCC in Chinese population [26]. However, the effect of SNP and HBV mutation interactions on the risk of HCC was not investigated in this study. In the current study, we investigated the contribution of rs3783553 and its interactions with HBV mutations in HCC development. The results of our study might be helpful in understanding the complex host-virus interaction in the development of HCC and in identifying HBV-infected subjects who are more likely to develop HCC.

Materials and methods

Study population

The current study included six groups of previously described participants [27]. Briefly, Group A comprised 1012 healthy controls who were free of serological HBV and HCV parameters, including antibodies to HBc (anti-HBc) and HCV (anti-HCV), and without history of liver diseases. They were recruited from the Health Examination Center at Changhai Hospital of the Second Military Medical University from September 2009 to June 2010. Group B comprised 302 HBV natural clearance participants who reported no history of HBV vaccination and were seronegative for HBsAg, HBV DNA, and anti-HCV, but seropositive for anti-HCV and anti-HBc. Group C comprised 316 asymptomatic HBsAg carriers (ASCs) who were seropositive for HBsAg but free of any clinical liver disease and had normal alanine aminotransferase (ALT) level (<40 U/L). Similar to group C, group B was free of clinical liver diseases. Both groups were initially recruited from our HBV-infected subjects cohort established in Yangpu District of Shanghai during the initial screening of seropositivity for HBsAg in 2010. These groups were revisited during the follow-up from June to December 2011. We only enrolled subjects who yielded a 100% concordance with the previous results during follow-up examinations. Groups D [316 patients with chronic hepatitis B (CHB)], E [358 HBV-infected patients with liver cirrhosis (LC)], and F [1021 HBV-infected patients with HCC] were recruited from Changzheng Hospital (361 cases), Changhai Hospital (269 cases), and Eastern Hepatobiliary Surgery Hospital (321 cases) of this university, South-west Hospital in Chongqing (23 cases), and the 88th hospital in Taian City, Shandong (47 cases), China from October 2009 to September 2011. CHB, LC, and HCC were diagnosed according to previously described criteria [11]. Patients who were seropositive for antibodies to HCV, hepatitis δ virus (HDV), or human immunodeficiency virus were excluded. All participants were of Han Chinese ancestry. The study protocol is in accordance with the Declaration of Helsinki (2000) and was approved by the Ethics Committee of the Second Military Medical University. All participants provided written informed consent.

Serological viral marker examination, HBV genotyping, and viral mutation analysis

Serological testing for HBV markers, α-fetoprotein, ALT, and viral load was conducted as previously described [11,28,29]. HBV genotyping, PCR amplification of HBV EnhII/BCP/PC region and preS region, and viral mutation analysis were conducted according to previous protocols [11,12].

DNA extraction and SNP genotyping

QIAquick PCR purification kits (QIAGEN, Germany) were used to extract genomic DNA from blood samples. Genotyping was conducted using fluorescent-probe real-time quantitative PCR (qPCR) in a LightCyclerTM480 (Roche, Basel, Switzerland). Primers and probes (Minor Groove Binder [MGB]) were designed by GeneCore Bio Technologies Co. Ltd. (Shanghai, China). The primer sequences were 5′-TTTGACTCTTTTGCCATTAAACTTACC-3′ (forward) and 5′-TGGTCTCATGGTTGTCAAAGTTG-3′ (reverse). The probes were FAM-TGTTTCATTCAATTCC-MGB and HEX-TCTTGTTTCAATTCCACCTG-MGB. Genotyping was performed for all participants. Laboratory technicians were blinded to case-control status. For quality control, blind duplicates (10% of the samples) were included to assess laboratory reliability, and 100% concordance rate was achieved.

Statistical analysis

Χ²- test was used to compare categorical variables among different groups. The levels of HBV DNA and ALT were transformed to normal distribution by logarithmic function. Student’s t-test or ANOVA was used to evaluate continuous variables. Hardy-Weinberg equilibrium (HWE) was tested with the exact test. Unconditional logistic regression was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) after age and gender adjustments. Gender may be a possible confounder. Thus, we further stratified the study population into males and females and evaluated the association within each stratum. We assessed the dominant [(homozygotes of insertion allele+ heterozygotes) vs. homozygotes of deletion allele] and recessive [homozygotes of insertion allele vs. (heterozygotes+ homozygotes of deletion allele)] model. Multiplicative interaction of SNP rs3783553 and HCC-related HBV mutations in HCC risk was evaluated separately in females and males, using multivariate regression analyses after adjusting for age and HBV genotype. All significance tests were two-sided. P<0.05 was considered statistically significant. All analyses were conducted using SPSS 16.0 for Windows (SPSS, Chicago, IL).

Results

Population characteristics

The demographic characteristics of the study participants are shown in Table 1. Briefly, HBV-infected participants (groups C, D, E, and F) were on average 10 years younger than healthy controls (group A) and HBsAg seroclearance participants (group B). The HCC patients had a higher proportion (84.1%) of males compared with the healthy controls (75.4%), HBsAg seroclearance participants (56.0%), and the HBsAg-positive individuals without HCC. Infection with HBV genotype C and HBeAg seroconversion were more common in the patients with HCC than in the HBsAg-positive individuals without HCC.

Associations of rs3783553 with the risks of HCC and other HBV-related clinical features

The genotyping success rates were 99.7%, 99.7%, and 99.5% for the healthy controls, HBV natural clearance participants, and chronic HBV-infected participants, respectively. rs3783553 was in HWE in the healthy controls (P = 0.42), HBsAg seroclearance individuals (P = 0.91), HBsAg-positive individuals without HCC (P = 0.54), and HCC patients (P = 0.80). Table 2 presents the genotype distributions of this SNP among different groups and the associations of rs3783553 with the risk of HCC. In general, no significant results were detected in the overall population or after stratifying by gender, either in the dominant or recessive model. We further investigated the association of this SNP with HCC risk in the participants infected with HBV genotypes B and C, respectively. No significant associations were discovered in either group (data not shown). Table 3 shows the association of rs3783553 with other HBV-related clinical features. Under the recessive model, the homozygotes of the TTCA insertion allele were associated with an increased risk of developing LC compared with the heterozygotes and homozygotes of the deletion allele combined. However, this association was not significant after stratifying by gender.

Associations of rs3783553 with HCC-related HBV mutations

HBV EnhII/BCP/PC region and the preS region were successfully sequenced from 57.7% and 47.1% of the HBsAg-positive subjects, respectively. Our previous study has identified the HBV mutations that are associated with risk of HCC [28,29]. In the current study, stepwise multivariate logistic regression analyses were used to assess the factors, including age, gender, rs3783553 genotype [(ins/ins+ del/ins) versus (del/del), indicating dominant model], and selected HBV mutations with risk of HCC. The HBV mutations T1674C/G, A1762T/G1764A, G1896A, and T1753V in the EnhII/BCP/PC region and preS deletion, preS2 start codon mutation, C2875A, C76A, and C7A in the preS region, rather than the rs3783553 genotype, were significantly associated with an increased risk of HCC (Table 4). We then evaluated the associations of SNP rs3783553 with the frequencies of these HBV mutations using the data of HBsAg-positive participants, including the HCC patients. The TTCA insertion allele was significantly associated with an increased frequency of HBV C7A mutation compared with homozygous TTCA deletion carriers [(del/ins+ ins/ins) versus del/del, adjusted OR= 1.48, 95% CI= 1.09-2.02, P = 0.013].

Multiplicative interactions of rs3783553 with the HBV mutations and their associations with HCC

We tested the multiplicative interactions of rs3783553 with all the significant HBV mutations (Table 4) in males and females separately. The interaction of rs3783553 with T1674C/G increased risk of HCC in females (OR= 2.23, 95% CI= 1.10-4.53). However, the association was not significant after age and HBV genotype were adjusted (Table 5). The interaction of rs3783553 with preS deletion significantly decreased risk of HCC in males after age and HBV genotype were adjusted (OR= 0.64, 95% CI= 0.42-0.98; P = 0.041) (Table 6).

Discussion

In this study, we did not find a significant association between rs3783553 and risk of HCC among the healthy controls, HBsAg seroclearance subjects, or HBsAg-positive subjects with or without liver diseases (Table 2). These data were inconsistent with the previous report that the rs3783553 variant was significantly associated with a decreased risk of HCC [26]. This inconsistency might have been caused by different controls in the two studies. Identifying the status of the controls is important to explore genetic susceptibility to HBV-related HCC in case-control studies. Recent genome-wide association studies have also reported contrasting results in genetic susceptibility to HBV-induced HCC [30-33]. The reasons may be complex, but we believe that HBV viral factors are the major confounders that affect the discovery of HCC genetic susceptible loci. The sizes of the effects (e.g., OR values in case-control studies) of genetic polymorphisms on HCC risk are usually smaller than those of the HBV mutations or genotypes [27,28,34,35]. The effect of genetic polymorphisms on risk of HCC can be greatly masked by the HBV mutations, whereas the effect of HBV mutations on risk of HCC can be solely significant in subjects with certain genetic background [27,28].

In the present study, the rs3783553 TTCA insertion allele was significantly associated with an increased frequency of HBV C7A mutation. HBV C7A mutation is extremely higher in HBV-infected subjects with liver diseases (CHB, LC, and HCC) than in those with ASCs [12]. C7A was more frequent in the subjects with rs3783553 TTCA insertion allele than those with the deletion allele. This result indicates that the rs3783553 genetic variant facilitates immuno-selection of this HBV mutation during the evolutionary process. The effect of viral factors and host proinflammatory molecules on tumor-promoting inflammation and anti-tumor immunity is complex. The interaction of viral mutations with genetic polymorphisms of some inflammatory molecules may contribute to the risk of HCC.

We found that multiplicative interaction of rs3783553 and preS deletion reduced the risk of HCC in males after age and HBV genotype were adjusted (Table 6). The interaction of rs3783553 with T1674C/G increased the risk of HCC in females (Table 5), and the interaction of rs3783553 with preS deletion reduced the risk of HCC in females (Table 6). However, the sample sizes became very small after the stratification, and the statistical power was low. We hypothesize that the variant genotype (TTCA insertion) of rs3783553 significantly decreases the influence of HBV preS deletion in promoting hepatocarcinogenesis. HBV preS deletion is a strong HCC-risk HBV mutation [9,10,12]. rs3783553 is an insertion/deletion (insertion or deletion of TTCA bases) polymorphism that is located at the miR-122 binding site in the 3′UTR of IL-1A. The TTCA insertion allele of rs3783553, which is associated with high IL-1α expression in situ or in serum, has also been associated with reduced risks of HCC and nasopharyngeal carcinoma in Chinese populations [26,36]. IL-1 is a pleiotropic cytokine that primarily affects inflammatory and immune responses as well as induces the expression of several important pro-inflammatory genes [37]. IL-1α, which is locally expressed in macrophages, has a mediating function in chronic liver damage and inflammation to HCC and expression of IL-1α on malignant cells stimulates anti-tumor immunity [20,37]. MiR-122 is a liver-specific miRNA that may influence HCV replication by binding RNA folding or RNA accumulation into replication complexes. Therefore, miR-122 serves as a therapeutic target of HCV infection [21,38]. Recently, miR-122 has been linked to HBV infection. miR-122 levels, which may facilitate viral replication and persistence, are significantly decreased in HBV-infected patients [39]. HBV mRNA inhibits miR-122 production and promotes HCC by upregulating miR-122-targeted molecule pituitary tumor-transforming gene 1 binding factor [40]. HBV X protein binds peroxisome proliferator-activated receptor-γand inhibits miR-122 transcription [41]. MiR-122 inhibits IL-1α expression in a dose-dependent manner, especially in cells with rs3783553 TTCA deletion [26]. Downregulation of miR-122 in HBV-infected subjects also facilitates IL-1α expression. Thus, the variant genotype of rs3783553 might predispose the host to express IL-1α in resident macrophages in the liver upon HBV infection, upregulate immune response to HBV, and attenuate the carcinogenic effect of HBV preS deletion. Further functional studies are warranted to clarify the preventive effect of rs3783553 TTCA insertion allele on HBV preS deletion-promoted hepatocarcinogenesis.

The present study has several limitations that need to be addressed. First, the two HBV regions were not amplified from a fraction of the HBsAg-positive participants, so the sample size for subsequent analyses was reduced. Second, although we did not find any statistically significant association between rs3783553 and risk of HBV-related HCC, other genetic polymorphisms at the miR-122 binding-sites should be evaluated for predicting the risk of this inflammation-related malignancy. Third, our study design is cross-sectional case-control study in nature.

In summary, our study suggested that the miRNA-122 binding site polymorphism at the IL-1A gene (rs3783553) did not significantly affect the risk of HBV-related HCC. However, the variant allele of rs3783553 significantly decreased the cancer-promoting effect of HBV preS deletion in the HBV-infected subjects. Future prospective studies should be conducted to evaluate the interaction of IL-1A gene polymorphisms, such as rs3783553, with HBV mutations in HBV-induced hepatocarcinogenesis.

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