CD44, IL-33, and ST2 Gene Polymorphisms on Hepatocellular Carcinoma Susceptibility in the Chinese Population.

The interleukin- (IL-) 33/ST2 axis plays a pivotal role in tumorigenesis through influencing cancer stemness and other mechanisms. CD44 is one of the critical markers of hepatocellular carcinoma (HCC) among the cancer stem cells (CSCs). There is still a lack of CD44 gene single-nucleotide polymorphisms (SNPs) combined with IL-33/ST2 pathway single-nucleotide polymorphisms in HCC susceptibility analysis literature, although CD44 and IL-33/ST2 have been reported separately in human cancers. This study is aimed at investigating the relationship between CD44, IL-33, and ST2 SNPs and HCC susceptibility and clinicopathological features. We analyzed 565 HCC patients and 561 healthy controls in the Chinese population. The genes for CD44rs187115A>G, IL-33 rs1929992A>G, and ST2 rs3821204G>C were typed using the SNaPshot method. We found that the distribution frequencies of CD44 and ST2 alleles and genotypes in both the HCC case group and the control group were statistically significant (p < 0.05). The results showed that individuals carrying at least one G allele of the CD44 rs187115 gene were at a higher risk than the AA genotype carriers (p = 0.007, odds ratio (OR) = 1.429, 95% confidence interval (CI): 1.102-1.854). Similarly, individuals with at least one C allele of ST2 rs3821204 had a higher risk of HCC than those with GG genes (p ≤ 0.001, OR = 1.647, 95% CI: 1.296-2.093). Combining the haplotype analysis of the 3 loci suggested that CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204 are associated with the risk of HCC and could potentially serve as useful genetic markers for HCC in some populations of China.

1. Introduction

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. The annual global incidence of primary liver cancer is 841,000; the death rate is 782,000; and it is the second highest mortality rate among males [1]. The mortality rate of liver cancer in China is the second highest among malignant tumors (new liver cancer accounts for more than 50% of the world's total) and is following a trend of annual increase [2]. Surgical resection, liver transplantation, and tumor ablation are the potential curative therapies; however, these treatment options are only applicable to patients in the early stage of disease [3, 4]. Due to the highly malignant potential of HCC, diagnosis is not usually made until an advanced stage, at which point there are no effective therapies to be offered [5]. Therefore, it is of pressing importance to identify which genes are responsible for susceptibility to HCC. Single-nucleotide polymorphism (SNP), a well-defined molecular biomarker, has been widely applied in HCC susceptibility evaluation [6, 7].

Various factors, including viral infection and cirrhosis, may be involved in the development of HCC [8, 9]. Tumor stem cells (CSCs), a small number of stem cell-like cancer cell subsets in tumor tissues, are characterized by both cancer cells and stem cells and are decisive in the initiation of HCC formation, growth, and metastasis [10, 11]. The CSC marker CD44 is essential in several malignancies, including HCC, and is the primary adhesion molecule of the extracellular matrix, playing a pivotal role in tumor cell differentiation, invasion, and metastasis [12, 13]. Recent studies have suggested that in the development of HCC, CD44+ cells were entwined with the genetic processes involved in cancer invasion and metastasis [14, 15]. HCC was often detected following the onset of cirrhosis, and the majority of patients worldwide with HCC have underlying cirrhosis [16]. The IL-33 gene, located on chromosome 9 (9p24.1), binds to ST2 and forms a trimer with IL-1R accessory protein (IL-1RAcP), which recruits downstream signaling molecules through the Toll/IL-1R (TIR) domains of ST2. The signaling pathways of nuclear factor kappaB (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinase (MAPK) are then activated, and the subsequent upregulation of the gene expression of proinflammatory cytokines leads to hepatic fibrosis [17-19]. Although CD44 and IL-33/ST2 have been well documented in human cancer metastasis or prognosis, respectively, evidence of the CD44 gene SNPs combined with IL-33/ST2 pathway functional polymorphisms in HCC susceptibility and clinical characteristics is scarce. We aimed to investigate in this study the association of these 3 polymorphisms with demographics, etiology, clinical features, and susceptibility to HCC.

2. Materials and Methods

2.1. Subjects and Clinical Data

The participant cohort was continuously recruited from September 2016 to December 2018 at the Affiliated Tumor Hospital of Guangxi Medical University. All patients were newly diagnosed and pathologically confirmed as having HCC, according to the American Association for the Study of Liver Diseases guidelines [20]. Ultimately, the study included 565 participants in the case group, 487 males and 78 females, aged 10–89 years. The control group was matched for sex and age and included a total of 561 cases (male = 488, female = 73), aged 22–78 years. The healthy control group patients were free of hypertension, diabetes, dyslipidemia, liver diseases, and cancer. Demographic data included medical record number, gender, age, drinking history, smoking history, tumor stage, and related biochemical indicators. All participants signed a written consent form after being informed of the study details. The Judging Committee approved the study of the Affiliated Tumor Hospital of Guangxi Medical University.

2.2. DNA Extraction and Genotyping Assays

We collected 2 ml of fasting peripheral blood from each participant and placed it in ethylenediaminetetraacetic acid- (EDTA-) K2 anticoagulant tube. After thorough mixing, it was stored in a refrigerator at -80°C for DNA extraction from the genome. Genomic DNA was extracted from the 2 ml of peripheral blood using a commercial kit according to the manufacturer's instructions (Adelaide, Beijing, China) and stored at –80°C before genotyping. The genotyping of CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204 was performed using the SNaPshot method as previously described [21], and negative controls were used in each test to ensure accuracy of the genotype evaluation. The primers are listed in Table 1.

Table 1

SNP oligonucleotide sequences used for related gene genotyping.

SNP	PCR primer
CD44 rs187115	Forward: 5′-TCAGGCAGGAGGAATAGGACA-3′Reverse: 5′-CTCCTGCCCAATAAAGCCAA-3′
IL-33 rs1929992	Forward: 5′-TATGACACAGGACCCCGGAA -3′Reverse: 5′- GAAGTCATCATCAACTTGGAACCT-3′
ST2 rs3821204	Forward: 5′-GACTGTTCCTGTTTGCTGGGA-3′Reverse: 5′-TTGTTCACTTTACCACCCTCGC-3′

2.3. Statistical Analysis

First, we assessed whether genotype frequencies were in the Hardy-Weinberg equilibrium (HWE). The Pearson two-sided chi-squared (χ2) test was used to analyze the HWE law to confirm whether the population of the study samples was representative of the populace [22]. When a p > 0.05 value was observed, the samples were representative of the population. Then, the χ2 test was used to examine the difference in clinicopathological features between the case group and the control group. The distribution data of alleles and genotypes were compared using the χ2 test and logistic regression analysis, and the relative risk was expressed by odd ratios (OR) and their 95% confidence intervals (CI). Additionally, unconditional logistic regression was used to correct the effects of confounding factors such as gender, age, drinking history, and smoking history on OR values and 95% CI. The haploid model analysis of the gene interaction was performed using the SHEsis software (http://analysis.bio-x.cn/myAnalysis.php) [23]. eQTL are regions of the genome containing DNA sequence variants that influence the expression level of one or more genes. We further explored the effects of the three significant SNPs (CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204) on their gene expression by investigating a public database, GTEx portal (https://gtexportal.org/) [24]. Statistical analysis of the data was performed using the statistical software package SPSS 24.0 (SPSS Inc., IBM, Chicago, IL, USA), and the test was performed using a two-sided test with a test level of α = 0.05. We used the false positive reporting probability (FPRP) to evaluate meaningful findings. We set 0.2 as the FPRP threshold, and 0.1 as the prior probability to detect the preponderance ratio (OR) associated with genotypes and haplotypes in the study to be 0.67/1.50 (protection/risk effect). Only significant results with an FPRP value of < 0.2 would be considered a noteworthy finding.

3. Results

3.1. Demographic and Clinical Characteristics of Participants

Initially, 593 subjects were enrolled in the HCC group, 21 cases had no pathological reports, and 7 cases had other cancers, and thus 28 cases were excluded. Ultimately, the study included 565 patients in the HCC group (males = 487, females = 78, age 10–89 years) and 561 in the healthy control group (males = 488, females = 73, age 22–78 years). Briefly, male participants showed a higher prevalence of HCC than females (487 vs. 78). Metastasis was found in 85 patients (15.0%). Stage of HCC was staged A or B in 259 patients (45.8%) and stage C or D in 306 patients (54.2%). Moreover, there were no statistical differences in the distributions of age, gender, smoking status, and alcohol consumption between the 2 groups. Detailed demographic and clinical characteristics of the study participants, including age, gender, smoking and drinking status, Barcelona clinic liver cancer (BCLC) stage, and metastasis status, are provided in Table 2.

Table 2

General characteristics of HCC patients and the normal controls.

Characteristics	Cases (n = 565)	Controls (n = 561)	p value
Age (year)			0.546
Range	10-89	22-78
Mean	53.62	52.15
<40	95	102
41-50	133	152
51-60	186	176
>60	151	131
Gender			0.453
Male	487	488
Female	78	73
BMI (kg/m2)			0.406
<18.5	62	51
18.5-23.9	366	359
≥24	137	151
Smoking status			0.734
No	344	336
Yes	221	225
Alcohol drinker			0.113
No	374	396
Yes	191	165
HBV infection
HbsAg (−)	47	479
HbsAg (+)	497	75	≤0.001
Liver cirrhosis
Absent	487	491
Present	78	70	0.51
BCLC stage
A+B stage	259
C+D stage	306
Metastasis
No	480
Yes	85

3.2. Association between CD44 rs187115 and Susceptibility and Clinicopathological Parameters of HCC

In this study, the distribution of CD44 gene rs187115 in the HCC group (p = 0.58) and the control group (p = 0.74) was consistent with the HWE. The frequency distribution differences of the GG, GA, and AA genotypes in the 2 groups were statistically significant (p = 0.010). We used the AA genotype and A allele as a reference to analyze the risk of HCC. Logistic regression was used to adjust the impact of confounding factors such as gender and age and for calculating the OR value of rs187115 on the risk of HCC and 95% CI. Ultimately, it was found that compared with other genotype individuals, people carrying the homozygous GG genotype were 2.469 times more likely to develop HCC than those carrying the AA gene (p = 0.027, adjusted OR (ORadj) = 2.469, 95% CI: 1.110–5.492). Furthermore, individuals who carried the heterozygous AG genotype were 1.359 times more likely to develop HCC than individuals carrying the GG gene (p = 0.026, ORadj = 1.359, 95% CI: 1.039–1.778). Moreover, compared with the A allele, individuals carrying the G allele had a 1.423 times higher risk of HCC (p = 0.003, OR = 1.423, 95% CI: 1.131–1.792), suggesting that the G allele mutation was associated with an increased risk of HCC. The CD44 rs187115 polymorphism genotype and allele frequencies for the HCC and control groups are listed in Table 3. Also, we further studied the clinical status of CD44 rs187115 gene polymorphism in HCC to assess whether there was a difference in the distribution of the rs187115 genotype among clinical subgroups. The results showed that the distribution of AG and GG genotypes of rs187115 in the hepatic fibrosis subgroup was significantly different (p = 0.038) (Table 4).

Table 3

Distribution and genotype frequencies of related polymorphisms in the HCC group and the control group.

Parameter	Case, n (%)	Controls, n (%)	OR (95% CI)	p OR	ORadj (95% CI)	p ORadj
CD44 rs187115
All
AA	383 (67.8)	421 (75.0)	1.00		1.00
AG	162 (28.7)	131 (23.4)	1.359 (1.039-1.778)	0.025	1.359 (1.039-1.778)	0.026
GG	20 (3.5)	9 (1.6)	2.443 (1.099-5.430)	0.028	2.469 (1.110-5.492)	0.027
AG+GG	182 (32.2)	140 (25.0)	1.429 (1.101-1.855)	0.007	1.429 (1.102-1.854)	0.007
Alleles
A	928 (82.1)	973 (86.7)	1.00		1.00
G	202 (17.9)	149 (13.3)	1.421 (1.129-1.789)	0.003	1.423 (1.131-1.792)	0.003
IL-33 rs1929992
All
AA	169 (29.9)	157 (28.0)	1.00		1.00
GA	261 (46.2)	276 (49.2)	0.879 (0.667-1.157)	0.357	0.886 (0.672-1.168)	0.390
GG	135 (23.9)	128 (22.8)	0.980 (0.708-1.356)	0.902	0.980 (0.708-1.358)	0.906
AG+GG	396 (70.1)	404 (72.0)	0.911 (0.704-1.178)	0.476	0.916 (0.708-1.186)	0.506
Alleles
A	599 (53.0)	590 (52.6)	1.00		1.00
G	531 (47.0)	532 (47.4)	0.983 (0.833-1.160)	0.840	0.984 (0.834-1.161)	0.848
ST2 rs3821204
All
GG	198 (35.0)	264 (47.1)	1.00		1.00
CG	255 (45.2)	230 (41.0)	1.478 (1.144-1.910)	0.003	1.483 (1.147-1.916)	0.003
CC	112 (19.8)	67 (11.9)	2.229 (1.564-3.177)	≤0.001	2.208 (1.548-3.149)	≤0.001
CG+CC	367 (65.0)	297 (52.9)	1.648 (1.297-2.093)	≤0.001	1.647 (1.296-2.093)	≤0.001
Alleles
G	631 (56.0)	690 (61.5)	1.00		1.00
C	499 (43.0)	432 (38.5)	1.532 (1.290-1.820)	≤0.001	1.527 (1.285-1.813)	≤0.001

OR: odds ratio; ORadj: adjusted odds ratio; CI: confidence interval.

Table 4

Association of CD44 rs187115 genotype with clinical characteristics in HCC patients.

Characteristics	rs187115			p value	rs187115		p value	rs187115		p value
	AA	AG	GG		AG	GG		AA	AG+GG
Age (year)
<40	147	57	6		57	6		147	63
≥40	235	106	14	0.589	106	14	0.659	235	120	0.351
Gender
Male	55	20	3		20	3		55	23
Female	327	143	17	0.800	143	17	0.728	327	160	0.563
BMI (kg/m2)
<18.5	43	16	4		16	4		43	20
18.5-23.9	242	109	14		109	14		242	123
≥24	97	38	2		38	2		97	40
BCLC stage
A+B stage	211	83	11		83	11		211	94
C+D stage	171	80	9	0.634	80	9	0.730	171	89	0.373
Smoking status
No	230	100	12		100	12		230	112
Yes	152	63	8	0.973	63	8	0.907	152	71	0.840
Alcohol drinker
No	251	107	15		107	15		251	122
Yes	131	56	5	0.692	56	5	0.402	131	61	0.838
Metastasis
No	272	181	17		181	17		272	198
Yes	71	21	3	0.008	21	3	0.545	71	24	0.002
Liver cirrhosis
Absent	116	53	2		53	2		116	55
Present	266	110	18	0.117	110	18	0.038	266	128	0.955
HBV infection
HbsAg (−)	37	18	1		18	1		37	19
HbsAg (+)	339	141	18	0.675	141	18	0.419	339	159	0.754
HCV infection	6	4	1		4	1		6	5
AST
Negative	204	79	9		79	9		204	88
Positive	178	84	11	0.460	84	11	0.770	178	95	0.226
ALT
Negative	235	163	12		163	12		235	175
Positive	147	67	8	≤0.001	67	8	0.309	147	75	0.031
GGT
Negative	130	66	8		66	8		130	74
Positive	252	97	12	0.322	97	12	0.966	252	109	0.132
AFP
Negative	154	62	11		62	11		154	73
Positive	228	101	9	0.343	101	9	0.144	228	120	0.580

ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: γ-glutamyl transpeptidase; AFP: alpha fetoprotein.

3.3. Association between IL-33 rs1929992 and Susceptibility and Clinicopathological Parameters of HCC

The distribution of IL-33rs1929992 locus genotype in the HCC group (p = 0.084) and the control group (p = 0.750) was consistent with HWE, which indicates a good representation of the population. There was no significant difference in the frequency distribution of the GG, GA, and AA genotypes between the 2 groups (p = 0.597). Additionally, the results showed that rs1929992 genotyping of GA, G vector, and G allele was not associated with the risk of HCC (Table 3). Furthermore, there were no significant associations between IL-33 rs1929992 and any of the clinicopathological parameters (Table 5).

Table 5

Association of IL-33 rs1929992 genotype with clinical characteristics in HCC patients.

Characteristics	rs1929992			p value	rs1929992		p value	rs1929992		p value
	AA	GA	GG		GA	GG		AA	GA+GG
Age(year)
Range	13-83	10-89	24-87		10-89	24-87		13-83	10-89
Mean	52.6	52.1	54.3		52.1	54.3		52.6	53.1
<40	23	51	15		51	15		23	663
≥40	146	210	120	0.056	210	120	0.053	146	330	0.057
Gender
Male	138	233	116		233	116		138	349
Female	31	28	19	0.082	28	19	0.329	31	47	0.051
BMI (kg/m2)
<18.5	15	32	14		32	14		15	46
18.5-23.9	112	164	90		164	90		112	254
≥24.0	42	65	31	0.816	65	31	0.735	42	96	0.182
BCLC stage
A+B stage	90	145	71		145	71		90	216
C+D stage	79	116	64	0.821	116	64	0.575	79	180	0.778
Smoking status
No	98	166	80		166	80		98	246
Yes	71	95	55	0.460	95	55	0.453	71	150	0.357
Alcohol drinker
No	114	178	88		178	88		114	266
Yes	55	83	47	0.831	83	47	0.398	55	130	0.948
Metastasis
No	143	218	119		218	119		143	337
Yes	26	43	16	0.470	43	16	0.221	26	59	0.882
Family history of cancer
No	145	219	121		219	121		145	340
Yes	24	42	14	0.302	42	14	0.121	24	56	0.985
Liver cirrhosis
Absent	47	89	35		89	35		47	124
Present	122	172	100	0.173	172	100	0.096	122	272	0.404
HBV infection
HbsAg (−)	18	23	14		23	14		18	37
HbsAg (+)	146	225	128		225	128		146	353
HCV infection	5	3	3	0.684	3	3	0.792	5	6	0.379
AST
Negative	82	135	76		135	76		82	211
Positive	87	126	59	0.402	126	59	0.387	87	185	0.300
ALT
Negative	97	155	94		155	94		97	249
Positive	72	106	41	0.066	106	41	0.056	72	147	0.221
GGT
Negative	65	106	56		106	56		65	162
Positive	104	155	79	0.855	155	79	0.868	104	234	0.587
AFP
Negative	67	108	53		108	53		67	161
Positive	102	153	82	0.886	153	82	0.684	102	235	0.822

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyl transpeptidase; AFP, alpha fetoprotein.

3.4. Association between ST2 rs3821204 and Susceptibility and Clinicopathological Parameters of HCC

The observed genotype frequency of ST2 rs3821204 was consistent with the expected distribution of HWE in both the HCC group (p = 0.071) and the control group (p = 0.125). The frequency distribution of the GG, CG, and CC genotypes in the 2 groups was statistically significant (p ≤ 0.001). The risk of HCC development was analyzed using the AA genotype and the A allele as a reference. After adjusting for gender and age using logistic regression, we calculated the OR value and 95% CI of rs187115 locus for the risk of HCC. Finally, the results showed that individuals with rs3821204 CG+CC genotype were 1.647 times more at risk of developing HCC than those with the GG genotype (p ≤ 0.001, ORadj = 1.647, 95% CI: 1.296–2.093) (Table 3). Similarly, individuals carrying the homozygous CC genotype were 2.208 times more likely to develop HCC than individuals carrying the GG gene (p ≤ 0.001, ORadj = 2.208, 95% CI: 1.548–3.149). Furthermore, individuals who carried the heterozygous CG genotype were 1.483 times more likely to develop HCC than individuals carrying the GG gene (p = 0.003, ORadj = 1.483, 95% CI: 1.147–1.916). Moreover, compared with the G allele, individuals carrying the C allele had a 1.532 times higher risk of HCC (p ≤ 0.001, OR = 1.527, 95% CI: 1.285–1.813), suggesting that the C allele mutation was associated with an increased risk of HCC. Also, we further studied the clinical status of ST2 rs3821204 gene polymorphism in HCC; however, there was no significant difference in the distribution of the ST2 rs3821204 genotype in the clinical subgroup analysis (p > 0.05) (Table 6).

Table 6

Association of ST2 rs3821204 genotype with clinical characteristics in HCC patients.

Characteristics	rs3821204			p value	rs3821204		p value	rs3821204		p value
	GG	CG	CC		CG	CC		GG	CG+CC
Age(year)
Range	10-78	19-87	24-89		19-87	24-89		10-78	19-89
Mean	52.8	52.2	52.9		52.2	52.9		52.8	52.6
<40	28	36	15		36	15		28	51
>40	170	220	97	0.981	220	97	0.864	170	317	0.926
Gender
Male	176	219	92		219	92		176	311
Female	22	36	20	0.250	36	20	0.359	22	56	0.173
BMI (kg/m2)
<18.5	21	30	11		30	11		21	41
18.5-23.9	125	168	74		168	74		125	242
≥24.0	52	57	27	0.883	57	27	0.832	52	84	0.670
BCLC stage
A+B stage	110	131	64		131	64		110	195
C+D stage	88	124	48	0.501	124	48	0.308	88	172	0.582
Smoking status
No	123	152	68		152	68		123	220
Yes	75	103	44	0.863	103	44	0.842	75	147	0.613
Alcohol drinker
No	131	165	77		165	77		131	242
Yes	67	90	35	0.752	90	35	0.452	67	125	0.958
Metastasis
No	122	211	95		211	95		122	306
Yes	17	44	17	0.417	44	17	0.623	17	61	0.222
Family history of cancer
No	167	221	96		221	96		167	317
Yes	31	34	16	0.783	34	16	0.807	31	50	0.511
Liver cirrhosis
Absent	62	77	31		77	31		62	108
Present	136	178	81	0.798	178	81	0.626	136	259	0.641
HBV infection
HbsAg (−)	16	11	11		11	11		16	22
HbsAg (+)	179	239	99		239	99		179	338
HCV infection	3	5	2	0.304	5	2	0.144	3	7	0.617
AST
Negative	108	127	60		127	60		108	187
Positive	90	128	52	0.575	128	52	0.506	90	180	0.415
ALT
Negative	121	148	75		148	75		121	223
Positive	77	107	37	0.271	107	37	0.107	77	144	0.936
GGT
Negative	90	95	42		95	42		90	137
Positive	108	160	70	0.821	160	70	0.964	108	230	0.060
AFP
Negative	83	99	44		99	44		83	143
Positive	115	156	68	0.789	156	68	0.933	115	224	0.494

ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: γ-glutamyl transpeptidase; AFP: alpha fetoprotein.

3.5. Haplotype Frequencies

Results obtained from SHEsis software suggested that the 3 SNPs (CD44, IL-33, and ST2) were in a strong linkage disequilibrium. The risk of HCC was affected when cooccurrence of these polymorphisms was examined using logistic regression analysis. We found 3 haplotypes to be associated with the risk of HCC. Compared to participants with other haplotypes, carrying the rs187115-rs1929992-rs3821204 G-G-C haplotype increased the risk of HCC by 3.181 times (p ≤ 0.001, OR = 3.181, 95%CI = 1.664-6.082). The results of haplotype frequencies are summarized in Table 7, and Table 8 shows the FPRP values of significant results at different prior probability levels because their probability of being a false positive result was <20%.

Table 7

The association of haplotype of each gene with hepatocellular cancer risk.

Haplotype	Case (%) N = 565	Control (%) N = 561	p	OR (95% CI)
rs187115-rs1929992-rs3821204
AAC	227.91 (0.202)	168.09 (0.150)	0.001	1.434 (1.152, 1.785)
AAG	264.26 (0.234)	343.55 (0.306)	≤0.001	0.692 (0.574, 0.834)
AGC	168.53 (0.149)	149.51 (0.133)	0.279	1.140 (0.899, 1.446)
AGG	267.30 (0.237)	310.86 (0.277)	0.027	0.808 (0.669, 0.977)
GAC	44.16 (0.039)	34.13 (0.030)	0.261	1.296 (0.823, 2.042)
GAG	62.66 (0.055)	44.23 (0.039)	0.073	1.431 (0.965, 2.122)
GGC	38.40 (0.034)	12.27 (0.011)	≤0.001	3.181 (1.664, 6.082)
GGG	56.77 (0.050)	59.36 (0.053)	0.774	0.947 (0.652, 1.376)
rs187115, rs3821204
AC	396.22 (0.351)	318.60 (0.284)	≤0.001	1.362 (1.139, 1.627)
AG	531.77 (0.471)	653.40 (0.582)	≤0.001	0.638 (0.540, 0.753)
GC	82.77 (0.073)	45.40 (0.040)	≤0.001	1.874 (1.292, 2.719)
GG	119.23 (0.106)	104.60 (0.093)	0.330	1.147 (0.870, 1.513)
rs187115, rs1929992
AA	492.04 (0.435)	511.85 (0.456)	0.321	0.919 (0.779, 1.086)
AG	435.96 (0.386)	460.15 (0.410)	0.238	0.903 (0.763, 1.070)
GA	106.96 (0.095)	78.15 (0.070)	0.031	1.396 (1.030, 1.893)
GG	95.04 (0.084	71.85 (0.064)	0.069	1.342 (0.976, 1.845)
rs1929992, rs3821204
AC	271.98 (0.241)	200.95 (0.179)	0.003	1.453 (1.184, 1.783)
AG	327.02 (0.289)	389.05 (0.347)	0.003	0.767 (0.642, 0.917)
GC	207.02 (0.183)	163.05 (0.145)	0.015	1.319 (1.054, 1.651)
GG	323.98 (0.287)	368.95 (0.329)	0.030	0.820 (0.686, 0.981)

Table 8

False positive report probability values for associations between the risk of HCC and the frequency of genotypes and haplotypes of the CD44 gene and ST2 dene in an Chinese population.

Genotype/haplotype	OR (95% CI)	p value	Statistical powera	Prior probability
				0.25	0.1	0.01	0.001	0.0001
CD44 rs187115
AG/AA	1.359 (1.039-1.778)	0.025	0.764	0.091	0.232	0.796	0.971	0.997
GG/AA	2.443 (1.099-5.430)	0.028	0.116	0.429	0.693	0.961	0.996	1.000
AG+GG/AA	1.429 (1.101-1.855)	0.007	0.642	0.034	0.095	0.563	0.921	0.991
ST2 rs3821204
CG/GG	1.478 (1.144-1.910)	0.003	0.545	0.016	0.046	0.345	0.842	0.982
CC/GG	2.229 (1.564-3.177)	≤0.001	0.014	0.002	0.006	0.065	0.411	0.875
CG+CC/GG	1.648 (1.297-2.093)	≤0.001	0.220	0.001	0.002	0.020	0.168	0.668

aStatistical power was calculated using the number of observations in the subgroup and the OR and p values in this table.

3.6. Expression Quantitative Trait Loci

We further explored biological effects of the three significant SNPs (CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204) on their gene expression by investigating a public database (GTEx portal).We found that genotypes of these SNPs were not associated with their gene expression in liver tissue and whole blood cells.

4. Discussion

In this study, the results showed that the distribution frequency of the CD44 rs187115 allele and genotype in the HCC case group and the control group was statistically significant (p < 0.05), which was consistent with the results of Liu et al. in lung cancer and Winder et al. in gastric adenocarcinoma [25, 26]. Also, we further showed that there was a significant difference in the distribution of the rs187115 and GG genotypes in the liver fibrosis subgroup. CD44 belongs to the family of adhesion molecules and is also a hyaluronic acid receptor, which means it is mainly involved in the adhesion between cells and the interstitial matrix [27]. It was reported that CD44 promoted HCC CSC stemness by regulating natural killer (NK) sensitivity or the tyrosine-protein kinase-Met-class I phosphoinositide 3-kinase-protein kinase B (c-Met-PI3K-AKT) signaling cascade, leading to poor prognoses of cancer and chemoresistance [28, 29]. Additionally, CD44 is involved in the maintenance of CSCs in HCC, possibly through the PI3K/AKT/mTOR pathway and the NOTCH3 signaling pathway [30]. In some genes, an SNP located in a coding, promoter, or regulatory region, may have a certain function, whereas CD44 SNP rs187115 is located in the first intron of CD44 [31, 32]. Until this point, the regulatory mechanism of CD44 intron 1 has not been reported, but it has been found that the polymorphism of the CD44 rs187115 gene may act on chemical resistance and cellular stress response in a p53-dependent manner in HCC [33].

As a bifunctional factor, IL-33 has both transcription factors and cytokine activity [34]. There is a strong correlation between the level of IL-33 and the progression of disease in a variety of malignant epithelial tumors [19, 35]. Previous studies have shown that rs1929992 was associated with the risk of several autoimmune diseases. Since it is functional and has not reported its relationship to HCC, we studied whether it is associated with HCC risk [36-38]. However, the results showed that the difference in distribution frequency of rs1929992 locus alleles and genotypes in the HCC case group and control group was not statistically significant (p > 0.05), which was similar to the findings of Jafarzadeh et al. in breast cancer [39]. Some studies have reported overexpression of IL-33 in colorectal cancer [19, 40], while in HCC, inconsistent results were observed. Zhang et al. found that increased IL-33 protein levels were present in HCC patients' serum and liver tissue [41], whereas Bergis et al. did not find a significant difference in IL-33 serum levels between HCC patients and healthy controls [42]. Thus, further replication studies with larger sample sizes are needed to identify the relationship of rs1929992 to the risk of HCC.

Recent studies have also found that soluble ST2 levels were highly expressed in the liver, lung, and breast cancer; malignant glioma; and other tumor tissues; this was associated with the occurrence, invasion, and metastasis of tumors [43-45]. In the present study, we found that the frequency of the ST2 rs3821204 C allele was higher in the HCC group than in the healthy control group; our results were consistent with the findings of Wei et al. on Chinese HCC patients [46]. The ST2 rs3821204 is located in the three prime untranslated regions (3′UTR) of sST2 mRNA, and the CC genotype of rs3821204 is highly correlated with elevated plasma sST2 levels in vivo [47]. The ST2 rs3821204 CC genotype may contribute to hepatocarcinogenesis by enhancing ST2 production at the transcriptional and translational levels [46]. The physiological effect of ST2 gene rs3821204 polymorphism on HCC patients is mainly exerted by enhancing the synthesis of ST2 protein [46]. Additionally, studies have shown that ST2 deficiency could prevent tumor progression in a mouse model [48]. Based on the above mechanisms, individuals with rs3821204 are prone to develop HCC.

In this study, by performing a haplotype analysis, we found that CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204 have a combined effect on HCC susceptibility. By analyzing the reasons, the development of HCC was related to various genetic polymorphisms, and changes in multiple genes could cause genetic and molecular abnormalities [49, 50]. There are several studies related to the IL-33/ST2 axis and CSCs [51, 52]. Our previous research clarified that IL-33 binds to its receptor ST2 and induces phosphorylation of c-Jun N-terminal kinase activation (JNK), which leads to the expansion of colon cancer cell stemness [19]. Also, some researchers have found that IL-33 can promote the activation of p38, increasing the levels of liver CSC markers expression, and epithelial to mesenchymal transition- (EMT-) like changes [53]. We suppose that IL-33/ST2 may enhance CD44 expression to initiate HCC, but further experimentation is required to elucidate the mechanism.

In summary, our genetic results suggest an association between SNPs (CD44 rs187115, ST2 rs3821204) and the risk of HCC. The combination of CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204 might be used as a marker to identify a subgroup at higher risk of HCC among the Chinese population. Unfortunately, the signaling pathway for HCC development from CD44 to IL-33/ST2 axis has not been reported, and the small sample size of this study may limit the applicability of these results. Therefore, future research will be required, recruiting larger sample sizes, careful design, and more clinical information to identify risk factors for HCC development from CD44 rs187115, IL-33 rs1929992, and ST2 rs3821204 polymorphisms and the underlying biological mechanisms leading to HCC.