Background
Hepatitis B virus (HBV) infection is highly prevalent and one of the major causes of morbidity and death worldwide. Globally, two billion people have been reported to be infected with HBV, of which approximately one-fifth (about 350–400 million) are chronic carriers; further, tens of millions of new HBV cases occur annually [
1]. The outcome of chronic HBV infection is highly variable, ranging from an asymptomatic carrier state with a normal liver histology to persistent carriers with severe and chronic liver diseases such as cirrhosis and hepatocellular carcinoma (HCC) [
2]. HCC is the fifth most frequent cancer in men and the seventh in women worldwide [
3], and ranks as the second most common malignancy in China, especially in Southern Guangxi [
4]. Among individuals persistently infected with HBV, one to three in every ten will develop liver cirrhosis and HCC [
5]. Such highly variable outcomes may be generally attributed to viral factors such as HBV infection [
6] and environmental factors such as dietary aflatoxin B1 exposure [
7], along with an individual’s immunological status and genetic background [
8]. Further, according to Peng et al. [
9], the morbidity of HCC can be distinct in individuals with the same exposure, suggesting that host immunological status and genetic factors may play important roles in the development of HBV-related HCC.
Interleukin-18 (IL-18), a novel proinflammatory cytokine belonging to the IL-1 cytokine super-family, is mainly produced by activated macrophages and can mediate both innate and adaptive immunity [
10,
11]. It was initially recognized as an interferon-γ (IFN-γ)-inducing factor [
12] and has a wide range of functions, including induction of the synthesis of IFN-γ in T cells and natural killer cells through synergistic action with IL-12 and IL-10, respectively [
13‐
15], promotion of Th1-type immune responses, and enhancement of the proliferative response and cytokine production of activated T cells [
16]. Recently, this cytokine was found to play various roles in chronic inflammation [
17] and autoimmune diseases, as well as in numerous infectious diseases [
18‐
20]. Further, IL-18 has been linked to acute liver injury [
21]. As HCC is a typical inflammation-related cancer, these biologic effects of IL-18 might thus be implicated with HCC development, suggesting that abnormal expression of IL-18 could be associated with the pathogenesis of this disease.
The human
IL-
18 gene is located on chromosome 11q22.2-22.3 and consists of six exons and five introns [
22]. So far, three single nucleotide polymorphisms (SNPs) in the promoter region of the
IL-
18 gene have been elucidated, namely −656G/T (rs1946518), −607C/A (rs1946519), and −137G/C (rs187238) [
23]. A change from the C to the A allele at position −607C/A and a change from the G to the C allele at position −137G/C in the promoter region were predicted to be the nuclear factor binding sites for the cAMP responsive element binding protein and H4TF-1 nuclear factor, respectively. Furthermore, polymorphisms of the two sites have been associated with
IL-
18 gene promoter transcription activity, which can influence the expression of IL-18 and, potentially, of IFN-γ [
23,
24]; these allelic changes may be the underlying mechanism of
IL-
18 involvement in various diseases. To date,
IL-
18 polymorphisms have been proved associated with various diseases, including chronic HBV infection [
17], systemic lupus erythematosus [
25], breast cancer [
26], oral cancer [
27,
28], thyroid cancer [
29], colorectal cancer [
30], and bladder cancer [
31]. However, results focused on the effect of
IL-
18 polymorphisms or IL-18 expression on the risk of HBV-related HCC remains controversial [
32‐
34], and no much study has taken IL-18 serum levels into consideration. The present study was performed in order to further investigate the possible role of
IL-
18 −607C/A and −137G/C polymorphisms on the susceptibility to HBV-related HCC and their impact on serum IL-18 serum levels. This study compares
IL-
18 polymorphisms and IL-18 serum levels between HBV-related HCC patients and healthy controls from Guangxi, China, a region with a high HBV prevalence.
Discussion
HBV infection remains a public health problem worldwide, being endemic in some regions of the world, especially in developing countries [
36]. HBV can lead to severe liver diseases such as chronic hepatitis, cirrhosis, and HCC [
37]. HBV-infected individuals are at a higher risk of developing HCC [
38], and age, sex, alcohol consumption, and smoking status are well known risk factors contributing to HCC incidence [
39,
40]. However, these factors do not fully account for the underlying mechanisms in HCC development, indicating that the immune status of individuals may play pivotal roles in disease progression. Through its involvement in the pro-inflammatory cytokine network,
IL-
18 is an important mediator of innate and adaptive immunity. Polymorphisms of this gene have been reported to influence the expression of IL-18 and may further lead to an alteration in an individual’s immune status, thus increasing carriers’ susceptibility to HCC development. However, this hypothesis is only partially confirmed herein.
In the present study, the
IL-
18 −137G/C polymorphism was observed to be significantly associated with HCC. The GC genotype and C allele of this SNP were associated with a significantly decreased risk of HCC compared with the GG genotype and G allele. Similarly, the A
−607C
−137 haplotype was significantly associated with a lower risk of HCC. Our findings were consistent with those of previous studies on
IL-
18 polymorphisms and HCC risk [
32‐
34], all of which observed a significant relationship between the −137G/C polymorphism and HCC risk, with a high G allele frequency being associated with an increased risk of HCC and, conversely, a low C allele frequency being associated with a decreased risk of HCC. However, they also found a significantly increased frequency of the −607 C/A AA genotype in HCC patients (AC genotype in Kim et al.’s study [
32] ), while we observed a null association between them.
These controversial results may due to the underlying genetic differences between various populations. In a study investigating the relationship between HBV infection and
IL-
18 promoter polymorphisms among three minority populations in Yunnan province, China, the genotype and allele frequencies of all three SNPs in the
IL-
18 promoter (−607 C/A, −137G/C, and −656G/T) were found to have distinct distributions [
41]. Further, the authors observed a significant difference in HBV infection among the three minority populations, concluding that the difference in genetic background among the various ethnicities may be an important factor responsible for HBV infection susceptibility [
41]. The present study was carried out in the Guangxi district, which is also ethnically diverse, and our study population contains both Han and Zhuang ethnicity—this may be the major contributing factor to the inconsistency result found in −607C/A polymorphism.
With regard to the serum IL-18 levels, they were found significantly lower in HCC patients compared to healthy subjects in the present study, such result is controversial to many studies. For instance, researches by Tangkijvanich et al. [
42] and Mohran et al. [
43] showed that IL-18 levels in HCC patients were significant higher than those in healthy controls, the latter study further concluded that serum IL-18 level was a suitable diagnostic marker in HCV-related HCC patients. However, though Wen et al. [
44] also observed significant differences in the transcription and expression levels of IL-18 among different HBV infector groups; interestingly, the highest was found in the fulminant hepatitis group and the lowest in the asymptomatic carrier group, and no significant differences between the chronic hepatitis and normal control groups were observed. These results indicate that serum IL-18 levels may be distinct at different disease stages. On the other hand, as all patients in our study were HBV-related HCC, with hepatitis B surface antigen, HBV core antibody, hepatitis Be antigen, or hepatitis Be antibody seropositive for at least 6 months, the majority of them have gone through antiviral treatment; but, according to He et al. [
45], antiviral treatment (pegylated interferon alpha) can induce a marked decline in IL-18 and remission of hepatic inflammatory in HCV infectors. These may be the underlying factors for the inconsistent results observed herein.
Furthermore, no association between the two
IL-
18 SNPs and IL-18 serum levels were observed in our study, which is inconsistent with previous reports. In the study conducted by Giedraitis et al. [
23], three alleles from the
IL-
18 promoter region (at positions −656, −607, and −137) were cloned and transfected into a HeLa cell line, leading to a tenfold higher
IL-
18 gene fragment activity compared to the negative control. This indicated that all three polymorphisms had a clear promoting activity and were able to influence the expression of IL-18. However, Giedraitis et al. [
23] only conducted studies on gene expression level, which were not fully representative of the transcription levels and, thus, of secretion levels. Therefore, our study is the first to show that −607 and −137 polymorphisms are not associated with the expression of IL-18 in serum. Nevertheless, since serum IL-18 levels may be affected by various factors, some of these may mask the gene polymorphism effects.
In summary, the −137G/C polymorphism of the IL-18 gene was observed to be significantly correlated with HCC risk. The A−607C−137 haplotype of the −607C/A and −137G/C SNPs was also associated with a decreased risk of HCC. These results indicate that the −137G/C SNP in IL-18 may be a protective factor against HCC. Further, lower serum IL-18 levels were found in HCC patients, however, the IL-18 −607C/A and −137G/C polymorphisms were not associated with IL-18 serum concentration. Considering the limited study population included herein, additional studies with larger samples and detailed clinical data are warranted in various ethnic populations.