Association Between Polymorphisms of Interleukin 1 Family Genes and Hepatocellular Carcinoma.

BACKGROUND Hepatocellular carcinoma (HCC) is one of the most common malignancies occurring worldwide and is most frequent type of liver cancer. The risk for developing HCC increases with the severity of inflammation and fibrosis. The members of the interleukin-1 (IL-1) family are primarily proinflammatory cytokines due to their ability to stimulate the expression of genes associated with inflammation and autoimmune diseases. Several studies have suggested that some proinflammatory cytokines, such as the IL-1 family (IL-1α, IL-1β, and IL-1 receptor antagonist) are involved in the pathogenesis of HCC. MATERIAL AND METHODS This study aimed to determine whether polymorphisms in the IL-1 family of genes are associated with HCC. We analyzed 178 HCC patients and 397 controls to investigate the association between polymorphisms in IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1RA) genes and HCC in the Korean population. All subjects were genotyped for the selected SNPs in IL-1α, IL-1β, and IL-1RA genes by Golden-Gate SNP Genotyping Assay. RESULTS Statistical analysis revealed a significant association at IL-1β between HCC and controls. Three individual polymorphisms (rs1143633, rs3917356, and rs1143627) were found to be associated with HCC. The SNPs of IL-1b gene (rs1143633A>G and rs1143627T>C) protected against HCC in the dominant model (p=0.027, OR=0.59, 95% CI=0.37-0.94; p=0.019, OR=0.56, 95% CI=0.34-0.91). The SNP of IL-1β gene (rs3917356G>A) increased the risk of HCC in the recessive model (p<0.001, OR=2.58, 95% CI=1.53-4.33), whereas other SNPs in IL-1α and IL-1RA showed no significant association between HCC patients and controls. CONCLUSIONS These results suggest that IL-1β in the IL-1 family contributes to HCC susceptibility.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies occurring worldwide. Almost 90% of primary malignant hepatic tumors lead to liver cancer among adults [1].

The distribution of HCC varies across the world; the highest HCC incidence is in South-East Asia and Sub-Saharan Africa, where prevalence of chronic hepatitis B (HBV) and hepatitis C (HCV) virus infection are high. These viruses are the most frequent etiological factors of HCC [2-4]. Also, increased body mass index (BMI) and diabetes with subsequent development of non-alcoholic steatohepatitis are significant risk factors for HCC. Other non-viral causes of HCC include iron overload syndromes, alcohol drinking, smoking, oral contraceptive use, and aflatoxin exposure, which are common in the developing world [5]. Male sex is another risk factor for HCC, since men are more susceptible to diseases than women, and the imbalance in the ratio of males to females in a population varies from 2: 1 up to 4: 1 [6,7].

The risk for developing HCC increases with the severity of inflammation and fibrosis [8,9]. HCC develops slowly in a background of chronic inflammation triggered by exposure to infectious agents (e.g., HCV and HBV), toxic compounds (e.g., alcohol), or metabolic impairment. Although the molecular mechanism links that connect inflammation and cancer are not thoroughly understood, a key role for cytokines such as interleukin-6 (IL-6) and interleukin-1 (IL-1) in HCC has been established in experimental animal models [10].

IL-1α and IL-1β are members of the IL-1 family. These cytokines are pleiotropic cytokines and are synthesized by a variety of cell types, including activated macrophages, keratinocytes, stimulated B lymphocytes, and fibroblasts. These cytokines are involved in various immune responses, inflammatory processes, and hematopoiesis, and they increase the expression of adhesion factors on endothelial cells and enable transmigration of leukocytes. IL-1 increases angiogenesis and can promote tumor invasiveness and metastasis [11,12]. Also, IL-1α stimulates cytotoxic cells, and proteolytic activation of protease by neutrophils induced by the IL-1 family enhances angiogenesis and tumorigenesis [13].

The IL-1 family of genes encoding the 3 proteins, IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1 RA) were mapped to chromosome 2q13-21 [14]. Association polymorphisms of IL-1 family genes and development of gastric cancer [15,16], breast cancer [17,18], and gallbladder cancer [19] were reported.

Specific polymorphisms associated with susceptibility to various diseases have been studied with great interest. Among these polymorphisms, single-nucleotide polymorphisms (SNPs) are very common throughout the genome. Therefore, they are considered appropriate tools for identifying individual susceptibility to common disease.

Recently, associations of numerous functional gene polymorphisms of proinflammatory and profibrogenetic factors with HCC have been found. The objective of the present study was to determine whether polymorphisms in the IL-1 family genes are associated with HCC in the Korean population.

Material and Methods

Subjects and clinical data

We recruited 178 patients (150 males and 28 females) diagnosed with HCC at Keimyung University Dongsan Medical Center. Each patient was diagnosed by aspiration cytology or biopsy. The tumor-node-metastasis system was used to stage the tumors. Tumor metastasis and portal vein were determined by computed tomography and angiography, respectively. As a control group, we recruited 397 patients (166 males and 231 females) who had visited our Health Promotion Center for routine check-ups from September to December 2004. All control group subjects were free of hypertension, dyslipidemia, diabetes, and liver diseases. The average age of controls was 47.84±10.56 years old (Table 1). All study subjects were Korean and provided written informed consent. The study was approved by our Institutional Review Board (IRB No. 03-09).

Table 1

Demographics and clinical variables of hepatocellular carcinoma patients.

Variable		Total (n=178)
Age		59.5	(31–80)
Gender	Female	28	(15.7)
	Male	150	(84.3)
Cause	Hepatitis B virus	133	(74.7)
	Hepatitis C virus	15	(8.4)
	Alcoholic liver disease	21	(11.8)
	Autoimmune liver disease	2	(1.1)
Type	Diffuse	13	(7.3)
	Massive	42	(23.6)
	Massive + diffuse	1	(0.6)
	Massive + nodular	2	(1.1)
	Nodular	101	(56.7)
	Simple nodular	19	(10.7)
Metastasis	Yes	25	(14.0)
	No	152	(85.4)
Stage	I	18	(10.1)
	II	50	(28.1)
	III	50	(28.1)
	IV-A	34	(19.1)
	IV-B	25	(14.0)
Size (cm)	<5	101	(56.7)
	≥5	75	(42.1)
PVT	Negative	115	(64.6)
	Positive	62	(34.8)
Albumin (g/dL)		3.5	(1.7–5.1)
AST (U/L)		65.5	(21–923)
ALT (U/L)		40	(5–654)

Age, albumin, AST and ALT are shown as median (range); Gender, type, metastasis, stage, size, PVT (portal vein thrombosis) are shown as frequency (percentage).

SNPs selection and genotyping

Twenty-one SNPs in the IL-1 family (IL-1α, IL-1β, and IL-1RA) genes of proinflammatory cytokines were selected based on database searches () for this study. The IL-1 family of genes are located on chromosome 2q14. The 21 SNPs were selected in the IL-1 family genes based on heterozygosity (above 0.1). The selected SNPs of the IL-1 family genes were located on chromosome 2q14, which included IL-1α and IL-1β (2 in the exon region, 1 in the 5′near gene, and 6 in the intron region).

Peripheral blood samples were collected from each subject and then stored at −20°C. The genomic DNAs of subjects were extracted from the peripheral blood samples with a commercial DNA extraction kit (Macherey Nagel, Germany). Genotyping of all SNPs was carried out using Golden-Gate SNP Genotyping Assay.

Statistical analysis

First, we assessed whether genotype frequencies were in Hardy-Weinberg equilibrium. The results were considered statistically significant at p values less than 0.05. The allele frequencies and genotype distribution were compared between HCC patients and controls using the chi-square test. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for effect of genotypes and allele frequencies at the 5% level of significance. For the logistic regression analysis of genetic data, SNPStats and SPSS 17.0 were used [20]. Logistic regression analysis was performed to investigate the association between SNPs of the IL-1 family of genes and HCC adjusted by age and sex. A linkage disequilibrium (LD) block of polymorphisms was analyzed using Haploview 3.32 [20,21]. The haplotypes and their frequencies were calculated using the EM algorithm.

Results

Demographic and clinical characteristics of study subjects

Table 1 shows demographic and clinical characteristics of study subjects. Male subjects showed higher prevalence of HCC than females (150 vs. 28, age range 31–80 years). Type of tumor was diffuse in 13 patients (7.3%), massive in 42 patients (23.6%), massive and diffuse in 1 patient (0.6%), massive and nodular in 2 patients (1.1%), nodular in 101 patients (56.7%), and simple nodular in 19 patients (10.7%). Metastasis was found in 25 patients (14.0%), and portal vein involvement was found in 62 patients (34.8%). Stage of HCC was stage I in 18 patients (10.6%), stage II in 50 patients (28.1%), stage III in 50 patients (28.1%), stage IV-A in 34 patients (19.1%), and stage IV-B in 25 patients (14.0%). Tumor size was over 5 cm in 75 patients (42.1%). Albumin, AST, and ALT ranges were 1.7–5.1, 21–923, and 5–654, respectively. The allele frequency in subjects followed Hardy-Weinberg equilibrium (p>0.05).

Logistic regression analysis of IL-1 family genes SNPs between HCC and controls

Table 2 shows the genotype distributions in IL-1α gene in HCC patients and controls. All genotype distributions were consistent with the Hardy-Weinberg principle (p>0.05). There were no significant differences between HCC patients and controls in rs3783550, rs17561, rs1800587, or rs1800794 (Table 2).

Table 2

Logistic regression analysis and genotype frequency of polymorphisms in IL-1α gene between HCC and control.

SNP locus	HWE	Genotype	HCC	Control	Model	OR (95% CI)	P-value
			n=178	n=397
rs3783550Intron	0.51	C/C	67 (53.2%)	186 (50.1%)	Codominant	0.81 (0.55–1.21)	0.31
		A/C	51 (40.5%)	157 (42.3%)	Dominant	0.86 (0.52–1.43)	0.56
		A/A	8 (6.3%)	28 (7.5%)	Recessive	0.51 (0.19–1.38)	0.18
rs17561missense (Ser114Ala)	0.68	G/G	163 (92.6%)	350 (88.2%)	Codominant	0.50 (0.24–1.06)	0.06
		T/G	13 (7.4%)	45 (11.3%)	Dominant	0.52 (0.24–1.14)	0.10
		T/T	0 (0.0%)	2 (0.5%)	Recessive	0.00 (0.00–NA)	0.11
rs18005875′-UTR	0.12	C/C	162 (91.5%)	349 (87.9%)	Codominant	0.70 (0.35–1.39)	0.30
		T/C	14 (7.9%)	45 (11.3%)	Dominant	0.66 (0.31–1.39)	0.27
		T/T	1 (0.6%)	3 (0.8%)	Recessive	0.87 (0.06–12.77)	0.92
rs18007945′-nearGene	0.23	C/C	158 (91.9%)	350 (88.6%)	Codominant	0.69 (0.34–1.39)	0.29
		T/C	13 (7.6%)	43 (10.9%)	Dominant	0.64 (0.30–1.39)	0.25
		T/T	1 (0.6%)	2 (0.5%)	Recessive	0.94 (0.06–14.63)	0.97

HWE is Hardy-Weinberg equilibrium. Genotype distributions are shown as number (%). Odds ratio (OR), 95% confidence interval (CI), and p-values were from logistic regression analysis with codominant, dominant, and recessive models controlling age and gender as covariates. SNP is single nucleotide polymorphism. Total number of each SNP is different, because genotypes of some SNPs are unreadable.

Table 3 shows the genotype distributions in IL-1β in HCC patients and controls. Rs1143633A>G and rs1143627T>C showed protective effects in the dominant model (A/A vs. A/G + G/G; T/T vs. T/C + C/C) (p=0.027, OR=0.59, 95% CI=0.37–0.94; p=0.019, OR=0.56, 95% CI=0.34–0.91), and rs3917356G>A increased the risk of HCC in the recessive model (G/G + G/A vs. AA) (p<0.001, OR=2.58, 95% CI=1.53–4.33), whereas all other loci (rs1071676, rs1143637, rs1143634, rs3136558, and rs143630) showed no significant associations.

Table 3

Logistic regression analysis and genotype frequency of polymorphisms in IL-1β gene between HCC and control.

SNP locus	HWE	Genotype	HCC	Control	Model	OR (95% CI)	P-value
			n=178	n=397
rs10716763′-UTR	0.25	G/G	174 (97.8%)	376 (94.7%)	Codominant	0.32 (0.09–1.14)	0.06
		C/G	4 (2.2%)	20 (5.0%)	Dominant	0.32 (0.09–1.14)	0.06
		C/C	0 (0.0%)	1 (0.2%)	Recessive	0.00 (0.00–NA)	0.57
rs1143637intron	0.25	G/G	174 (97.8%)	376 (94.7%)	Codominant	0.32 (0.09–1.14)	0.06
		A/G	4 (2.2%)	20 (5.0%)	Dominant	0.32 (0.09–1.14)	0.06
		A/A	0 (0.0%)	1 (0.2%)	Recessive	0.00 (0.00–NA)	0.57
rs1143634synonymous (Phe105Phe)	0.23	C/C	174 (97.8%)	377 (95%)	Codominant	0.32 (0.09–1.14)	0.06
		T/C	4 (2.2%)	19 (4.8%)	Dominant	0.32 (0.09–1.14)	0.06
		T/T	0 (0.0%)	1 (0.2%)	Recessive	0.00 (0.00–NA)	0.57
rs1143633intron	0.72	A/A	71 (43.3%)	141 (35.9%)	Codominant	0.75 (0.53–1.06)	0.10
		A/G	73 (44.5%)	194 (49.4%)	Dominant	0.59 (0.37–0.94)	0.027
		G/G	20 (12.2%)	58 (14.8%)	Recessive	0.97 (0.50–1.91)	0.94
rs3136558intron	0.73	T/T	48 (28.1%)	118 (30.0%)	Codominant	1.18 (0.86–1.62)	0.31
		T/C	86 (50.3%)	190 (48.4%)	Dominant	1.23 (0.75–2.01)	0.41
		C/C	37 (21.6%)	85 (21.6%)	Recessive	1.26 (0.73–2.19)	0.41
rs1143630intron	0.26	C/C	117 (70.1%)	261 (66.2%)	Codominant	1.00 (0.66–1.51)	1.00
		A/C	42 (25.1%)	118 (29.9%)	Dominant	0.92 (0.57–1.50)	0.75
		A/A	8 (4.8%)	15 (3.8%)	Recessive	1.58 (0.50–4.95)	0.44
rs3917356intron	0.22	G/G	52 (32.1%)	121 (30.8%)	Codominant	1.30 (0.96–1.76)	0.09
		A/G	56 (34.6%)	183 (46.6%)	Dominant	0.83 (0.51–1.36)	0.47
		A/A	54 (33.3%)	89 (22.6%)	Recessive	2.58 (1.53–4.33)	<0.001
rs11436275′-UTR	0.48	T/T	56 (33.5%)	105 (26.6%)	Codominant	0.88 (0.65–1.20)	0.42
		T/C	61 (36.5%)	190 (48.1%)	Dominant	0.56 (0.34–0.91)	0.019
		C/C	50 (29.9%)	100 (25.3%)	Recessive	1.33 (0.80–2.23)	0.27

HWE is Hardy-Weinberg equilibrium. Genotype distributions are shown as number (%). Odds ratio (OR), 95% confidence interval (CI), and p-values were from logistic regression analysis with codominant, dominant, and recessive models controlling age and gender as covariates. SNP is single nucleotide polymorphism. Total number of each SNP is different, because genotypes of some SNPs are unreadable.

There were no significant differences among HCC patients and controls in IL-1RA gene polymorphism (rs2234679, rs928940, rs439154, rs419598, rs315952, and rs315951) (Table 4).

Table 4

Logistic regression analysis and genotype frequency of polymorphisms in IL-1RA gene between HCC and control.

SNP locus	HWE	Genotype	HCC	Control	Model	OR (95% CI)	P-value
			n=178	n=397
rs22346795′-UTR	0.79	G/G	148 (84.6%)	331 (83.6%)	Codominant	0.73 (0.40–1.34)	0.31
		C/G	27 (15.4%)	62 (15.7%)	Dominant	0.74 (0.40–1.35)	0.32
		C/C	0 (0.0%)	3 (0.8%)	Recessive	0.00 (0.00–NA)	0.56
rs928940intron	0.25	G/G	42 (34.1%)	114 (31.1%)	Codominant	0.95 (0.67–1.34)	0.76
		T/G	53 (43.1%)	170 (46.5%)	Dominant	0.84 (0.49–1.45)	0.54
		T/T	28 (22.8%)	82 (22.4%)	Recessive	1.05 (0.57–1.93)	0.89
rs4391545′nearGene	0.38	A/A	77 (47.8%)	166 (42.5%)	Codominant	0.81 (0.58–1.14)	0.22
		A/G	60 (37.3%)	171 (43.7%)	Dominant	0.66 (0.41–1.05)	0.07
		G/G	24 (14.9%)	54 (13.8%)	Recessive	1.04 (0.53–2.03)	0.90
rs419598synonymous (Ala39Ala)	0.72	T/T	155 (88.6%)	345 (87.1%)	Codominant	0.69 (0.35–1.37)	0.28
		T/C	20 (11.4%)	50 (12.6%)	Dominant	0.69 (0.35–1.37)	0.28
		C/C	0 (0.0%)	1 (0.2%)	Recessive	0.00 (0.00–NA)	0.76
rs315952synonymous (Ser112Ser)	0.67	C/C	48 (38.1%)	122 (33.5%)	Codominant	0.88 (0.62–1.25)	0.49
		T/C	55 (43.6%)	174 (47.8%)	Dominant	0.83 (0.49–1.41)	0.50
		T/T	23 (18.2%)	68 (18.7%)	Recessive	0.86 (0.45–1.65)	0.65
rs3159513′-UTR	0.17	C/C	74 (46.0%)	141 (36.4%)	Codominant	0.74 (0.54–1.01)	0.06
		G/C	61 (37.9%)	174 (45.0%)	Dominant	0.65 (0.41–1.03)	0.07
		G/G	26 (16.1%)	72 (18.6%)	Recessive	0.81 (0.51–1.28)	0.37

HWE is Hardy-Weinberg equilibrium. Genotype distributions are shown as number (%). Odds ratio (OR), 95% confidence interval (CI), and p-values were from logistic regression analysis with codominant, dominant, and recessive models controlling age and gender as covariates. SNP is single nucleotide polymorphism. Total number of each SNP is different, because genotypes of some SNPs are unreadable.

Logistic regression analysis of IL-1 family genes SNPs with clinical characteristics in HCC patients

Table 5 shows the relationship of SNP of IL-1β gene with clinical characteristics in males with HCC. Among tested SNPs of IL-1 family genes, rs3917356 showed a significant association with portal vein thrombosis (PVT) (OR=2.17, 95% CI=1.08–1.35, p=0.029) and metastasis (OR=2.70, 95% CI=1.09–6.67, p=0.032). However, all other loci did not show any significant associations with clinical characteristics in males with HCC.

Table 5

Relationship of SNP of IL-1β gene with clinical characteristics in male with HCC.

		rs3917356		OR (95% CI)	p
		G/G+G/A	A/A
Albumin (g/dL)	<3.5	43 (64.2)	24 (35.8)	1	0.97
	≥3.5	53 (63.9)	30 (36.1)	1.01 (0.52–1.98)
AST (U/L)	65.5	44 (63.8)	25 (36.2)	1	0.71
	≥65.5	52 (66.7)	26 (33.3)	0.88 (0.45–1.74)
ALT (U/L)	<40	44 (62.0)	27 (38.0)	1	0.62
	≥40	52 (65.8)	27 (34.2)	0.85 (0.43–1.65)
PVT	Negative	68 (70.8)	28 (29.2)	1	0.029
	Positive	28 (52.8)	25 (47.2)	2.17 (1.08–1.35)
Stage	I + II	31 (62.0)	19 (38.0)	1	0.75
	III + IV	64 (64.6)	35 (35.4)	0.89 (0.44–1.80)
Metastasis	Yes	85 (67.5)	41 (32.5)	1	0.032
	No	10 (43.5)	13 (56.5)	2.70 (1.09–6.67)
Cause	Hepatitis	78 (62.4)	47 (37.6)	1	0.51
	Alcohol	14 (70.0)	6 (30.0)	0.71 (0.26–1.87)
Size (cm)	<5	50 (61.7)	31 (38.3)	1	0.49
	≥5	45 (67.2)	22 (32.8)	0.79 (0.40–1.56)

Variables are shown as frequency (percentage). Albumin, AST, and ALT are divided by median value. Odds ratio (OR), 95% confidence interval (CI), and p-values were from logistic regression analysis controlling age as covariates.

Discussion

It is believed that immune system-mediated chronic inflammation of the liver can lead to HCC development because the former induces continuous cell death, resulting in cell proliferation and increased frequency of genetic alterations [22,23].

The proinflammatory cytokines of the IL-1 family, most notably IL-1β and IL-18, also have very important roles in antimicrobial host defense. IL-1a and IL-1β, which bind and activate the same receptor, 8630372, activate the release of other proinflammatory cytokines such as TNF and IL-6, and induce a Th (helper T cell) 17 bias in the cellular adaptive responses [24].

In vivo, IL-1 is largely responsible for the acute-phase response, which includes fever, acute protein synthesis, anorexia, and somnolence [25]. IL-18 is essential for the induction of interferon gamma (IFN-γ) and Th1 responses. Through these mechanisms, cytokines of the IL-1 family are a crucial component of host defense against infections.

Many proinflammatory cytokines, such as IL-1β and IFN-γ polymorphisms have been associated with an increased risk of HCC [26-28]. Many studies have been conducted to analyze SNPs as genetic markets as a result of their high density and even distribution in the human genome.

Here, we investigated whether the IL-1 family (IL-1α, IL-1β, and IL-1RA) of genes polymorphisms are related to risk of HCC by genotyping 21 selected SNPs in the Korean population. In this study, we found that 3 SNPs of IL-1β gene were association with HCC. In our investigation of the association between IL-1 family genes and HCC progression, only rs3917356 showed a significant association with portal vein thrombosis (OR=2.17, 95% CI=1.08–1.35, p=0.029) and metastasis (OR=2.70, 95% CI=1.09–6.67, p=0.032). However, all other loci did not show any significant associations with clinical characteristics in male HCC.

Rs1143633A>G and rs1143627T>C revealed protective effects in the dominant model (p=0.027, OR=0.59, 95% CI=0.37–0.94; p=0.019, OR=0.56, 95% CI=0.34–0.91), and rs3917356G>A was found to be a risk factor of HCC in the recessive model (p<0.001, OR=2.58, 95% CI=1.53–4.33). Rs1143633 and rs3917356 are located in an intron of the IL-1β gene and do not lead to an amino acid change in the IL-1β protein. However, rs1143627 is associated with the transcription of IL-1β [29].

It is an attractive hypothesis that the IL-1β rs1143627 T allele enhances IL-1 production in the liver and induces injury to hepatocytes, which may result in the development of HCC. In this study, rs1143627 T allele and C allele were found to be risk and protective factor, respectively, and this result corresponds with the previous hypothesis. In another study, IL-1β gene was reported to be a possible candidate gene for increased risk of persistent HCV and HBV infection and fibrosis. IL-1β rs16944 C allele is a genetic marker for HCC development in chronic hepatitis B patients in the Thai population, with the possibility of an association with high IL-1β production in the liver [30], and rs1143627 T allele or rs16944/rs1143627 haplotype C-T is associated with the presence of HCC with chronic HCV infection in the Japanese population. The IL-1β rs1143627 T allele and MMP-3 5A allele are cooperative risk factors for poor prognosis in HCC patients, suggesting that these gene polymorphisms are potential markers for predicting the prognosis of HCC patients [31].

Conclusions

Our genetics results suggest an association between SNPs (rs1143627, rs1143633, rs3917356) of IL-1β gene in the IL-1 family and the risk of HCC in the Korean population. These SNPs of IL-1β gene might be used as a marker to identify a subgroup at higher risk of HCC in Korean, although further research is necessary to explain the relationship between the expression levels of IL-1α, IL-1β, and IL-1RA and SNPs of IL-1β gene in HCC patients.