Association of CD44 polymorphisms and susceptibility to HBV‐related hepatocellular carcinoma in the Chinese population

Abstract Background This study aimed to determine whether CD44 polymorphisms were correlated with hepatocellular carcinoma (HCC) and to reveal a new potential target for early prediction, prevention, and diagnosis of HCC. Method This study involved 96 cases with chronic hepatitis B (CHB), 96 cases with hepatitis B virus‐related liver cirrhosis (LC), 204 cases with HCC related to the hepatitis B virus, and 210 healthy controls. The genotype of rs8193 was determined using the restriction fragment length polymorphism method, while the genotypes of rs10836347 and rs13347 were determined by direct sequencing. Results The results showed that patients with the CD44 rs13347 TT and T allele polymorphisms exhibited higher risks of LC than those carrying the CC genotype and C allele. The CD44 rs13347 CT and TT genotypes and T allele were significantly associated with an increased risk of HCC after adjusting for gender, age, smoking, and alcohol consumption (for CT: odds ratio [OR] = 1.626, 95% confidence interval [CI] = 1.057‐2.500, P = .027; for TT: OR = 1.965, 95% CI = 1.043‐3.702, P = .037; and for T: OR = 1.461, 95% CI = 1.091‐1.956, P = .011). In the rs13347 site of the female population, the CT and TT genotypes were related to the high occurrence of HCC. In the population aged ≥50 years, carriers of the CD44 rs13347 CT and TT alleles were more susceptible to HCC compared with CC carriers. Those who consumed alcohol who carried the rs10836347 CT genotype exhibited a risk factor for HCC. Conclusion For the CD44 rs13347 site, mutations in the T allele might be a risk factor for HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is one of the most frequent and fatal cancers. It is more common in less developed countries, but its incidence in the Western world is showing an alarming rise. The latest research reported 841 080 new cases of and 781 631 deaths from liver cancer in 2018. 1 With its high morbidity and mortality rates of HCC, China accounted for about half of the total cases and deaths of HCC. 1 In fact, Chen et al 2 estimated that HCC had become the third major cause of cancer-related death in China. Over the past few decades, surgical resection and a variety of comprehensive treatments have been used to treat HCC, but due to its high degree of malignancy, the prognosis is poor, and long-term survival rates remain low. Therefore, the effective early detection, diagnosis, and treatment of HCC have become urgent.
The pathogenesis of a tumor is a multistep, multifactored, complicated process. 3 Several studies have confirmed that environmental risk factors for HCC include infection with the hepatitis B virus (HBV) or hepatitis C virus (HCV), aflatoxin intake, alcohol use, and exposure to carcinogens. 4,5 However, under the same environmental conditions, some people develop HCC and some do not, suggesting that genetic factors influence HCC's development.
CD44 is a major cell adhesion molecule of an extracellular matrix and has been observed in various cells and tissues. It is involved in many physiological processes, including cell proliferation, angiogenesis, invasion, and metastasis. 6 The CD44 gene is located on chromosome 11 in humans and contains 20 exons, including 10 constant exons and 10 variant exons. Depending on whether the exons are involved in transcription and alternative splicing, CD44 is divided into a standard form (CD44s) and splice variant isoforms of the protein (CD44v). 7 Several reports have documented that altered CD44 expression and the interaction of hyaluronan and CD44 may regulate cell growth, survival, invasion, and metastasis in various cancers. [8][9][10] Chou et al 11 identified that rs1425802, rs713330, rs11821102, rs10836347, and rs13347 polymorphisms of the CD44 gene were not correlated with risk of HCC. However, patients with the CD44 polymorphism rs187115 had higher risks of HCC. Similarly, Chen et al 12 reported that rs187115 variant carriers with the G allele genotypes had an increased risk of HCC.
However, few studies have investigated the relationship between CD44 polymorphisms and HCC. Moreover, the regulation of CD44 expression in HCC is not completely understood. Therefore, this study thoroughly investigated the association between CD44 polymorphisms to elucidate a more exact relationship between CD44 polymorphisms and the risk of HCC. The study further tried to reveal a new potential target for the early prediction, prevention, and diagnosis of HCC. The criteria for CHB included having abnormal levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and simultaneously being persistent HBV surface antigen-positive for more than half a year. HBV-related LC was defined as being HBV surface antigen-positive, in which the liver pathology showed clinical manifestations of portal hypertension and in which ultrasonography showed evidence of cirrhosis. HCC patients were confirmed by imaging examinations, including ultrasonography, magnetic resonance imaging, or computed tomography. Additionally, the a-fetoprotein (AFP) elevation of these patients was higher than the cutoff value of >400 ng/mL. Healthy controls were included who had previously been examined and had no tumor, who were HBsAg-negative, and who had normal liver function and other laboratory tests. They had no family history of tumors or major disease. Nonsmokers and nondrinkers were defined as those who had never smoked or consumed alcohol. Questionnaires included demographic information such as sex, age, and smoking and drinking habits, and informed consent was collected from each enrolled participant. The study approval was obtained from the ethics committee of the First Affiliated Hospital of Guangxi Medical University.

| Selection of CD44 polymorphisms
A total of three single nucleotide polymorphisms (SNPs) in CD44 were selected for this study. Rs8193 was selected since the gene polymorphisms of this SNP have been found to be associated with colon cancer recurrence and gastric cancer. 8,9 The rs10836347 and rs13347 SNPs located in the 3′ untranslated region (3′ UTR) and the T allele mutations in the CD44 rs13347 site were chosen because they affect gene transcriptional activity. 10

| CD44 genotyping
Genomic DNA was isolated from fresh blood samples from each patient using the QIAamp DNA Blood Mini Kit according to the manufacturer's instructions. The upstream and downstream primers of rs8193, rs10836347, and rs13347 were designed and synthesized by the Shanghai Sangon Biotech Company. Since rs10836347 and rs13347 were close to each other, the two sites were combined to design primers, and the genotypes of the two sites could be determined by sequencing. The genotyping of rs8193 was performed using the restriction fragment length polymorphism method. The sequence of primers used in the polymerase chain reaction (PCR) and the essential reaction conditions are shown in Table 1.

| Statistical analysis
The genotype and allele frequencies of SNPs in the four groups were calculated using the direct calculation method. Demographic and clinical data among the groups were analyzed using the ANOVA method. The SNPs were analyzed for deviation from the Hardy-Weinberg equilibrium (HWE) using the chi-square test. Binary logistic regression was performed to determine the odds ratio (OR), and four factors (gender, age, smoking, and alcohol consumption) were adjusted. Stratified analyses were assessed according to gender, age, smoking, and alcohol consumption. All data were analyzed with SPSS 13.0. When the P value was less than .05, the results were considered significant.

| Characteristics of study populations
The features of the three disease groups and controls included in this study are listed in Table 2. Significant statistical difference was found regarding the age (all P < .001), while the proportions of the sexes in the four groups were consistent (P = .167, .327 and .053).
The smoking and drinking status of the patients was taken into account with respect to its influence on the association between CD44 polymorphisms and HCC. For smoking, a significant difference was found between controls and HBV-related liver cirrhosis (P = .005).
For drinking, a significant differences were found between controls and chronic hepatitis B (P = .036) and HCC (P = .009).
Tests for the HWE were performed separately for all SNPs in the healthy controls and then for the three disease groups; the observed genotype frequencies were both in agreement with the HWE (all P > .05).

| Alleles and genotype distributions of CD44 polymorphisms
The allele and genotype distributions of CD44 rs8193, rs10836347, and rs13347 among the disease groups and the controls are shown in Table 3. Binary logistic regression analysis for the CD44 rs8193 polymorphism (after adjusting for sex, age, smoking, and alcohol consumption) revealed that there were no statistically significant differences in allele and genotype distribution between cases and controls. The results showed that the CD44 rs8193 polymorphisms had no relationship with the risk of CHB, LC, or HCC in any genetic models. Additionally, when the rs10836347 site was analyzed, no significant difference was found between the controls and the cases (Table 3).
Nonsignificant associations with CHB risk were suggested for CD44 rs13347 when the healthy controls were compared with the CHB cases (Table 3). However, patients with the CD44 rs13347 TT and T allele polymorphisms exhibited 2.601-fold (95% CI = 1.242-5.447, P = .011) and 1.588-fold (95% CI = 1.102-2.288, P = .013) higher risks of LC, respectively, than those carrying the CC genotype and C allele. Similar results were found in the recessive analytic model; carriers of the TT genotype for CD44 rs13347 had a twofold increased risk of LC (95% CI = 1.162-4.568, P = .017). After TA B L E 1 Primer sequence and the reaction condition for genotyping CD44 polymorphisms  (Table 5). Whether the person was a smoker or nonsmoker, no evidence of an association between CD44 polymorphisms and HCC susceptibility was found (Table 6).

| Haplotype analyses of CD44 SNPs and HCC risk
Haplotype analyses were further performed in HCC patients and healthy controls using the SHEsis software. Four possible haplotypes (CCC, CCT, TCC, and TCT) were derived from the observed genotypes, and their distributions in both groups are shown in

| D ISCUSS I ON
CD44 is a multifunctional, transmembrane glycoprotein that was first defined as a lymphocyte homing receptor expressed in embryonic stem cells, hematopoietic stem cells, and tumor stem cells.
CD44 is related to a series of basic biological processes, including  The results from a study by Liu et al 19 showed that rs187115 sites of the G allele significantly increased the risk of non-small-cell lung cancer (NSCLC) compared with the AA genotype. They also found that the tumor grade of AG and GG gene carriers was higher and were more prone to bone metastases. However, the researchers found no association between rs13347 and NSCLC risk.
Other studies have also demonstrated that the CD44 gene has a role in the regulation of tumor metastasis and that a high expression of CD44 is usually present in metastatic tumors, especially in tumors with higher bone metastasis. 20 Data from the literature have also indicated that excessive alcohol consumption is an important risk factor for HCC; in the present study, the stratified analysis found that the rs10836347 CT genotype and the rs13347 TT genotype were correlated with a higher risk of HCC in those who consumed alcohol, suggesting that those two genotypes might be risk factors for HCC. Interactions between osteopontin (OPN) and CD44 have been reported to inhibit the expression of the IL-10 Th2 cytokine; additionally, these interactions have been shown to be involved in inflammatory response. 25 Long-term alcohol usage may induce liver cell injury and cause a sustained immune response, which might explain the possible role of the CD44 polymorphisms in HCC. 25 However, a few limitations remained in this study. First, the total sample size was relatively small, especially for the HBV-related LC subgroup. Second, only three SNPs were selected; more SNPs should be studied. Third, this case-control study only involved the Chinese population of the Guangxi province. Therefore, further well-designed studies are demanded from other regions of China.
Finally, a gene expression functional assay was not taken; this should be taken in the future.

| CON CLUS ION
In conclusion, data from this study indicated that CD44 rs13347 polymorphisms were associated with the risk of HCC, and mutations in the T allele might be a risk factor for HCC.

ACK N OWLED G M ENTS
We thank all of the fellows for many useful suggestions.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.