Abstract
Aims: Our study aimed to investigate the association of cytotoxic T-lymphocyte antigen-4 (CTLA-4) rs231775 polymorphism with hepatocellular carcinoma (HCC) susceptibility. Methods: Genotypes distribution of the control was tested by Hardy-Weinberg Equilibrium (HWE). CTLA-4 rs231775 polymorphism was analyzed in 80 patients with HCC and 78 healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, and the expression level of CTLA-4 in the serum of all subjects was detected using enzyme linked immunosorbent assay (ELISA) kit. Odd ratio (OR) with 95% confidence interval (CI) were calculated by chi-squared test to determine the correlation of CTLA-4 rs231775 polymorphism and the risk of HCC. Results: The genotypes frequencies of the control group were in accordance with HWE. The frequencies of genotype AA and allele A in CTLA-4 rs231775 polymorphism were significantly higher in cases than the control group (AA vs. GG: OR=2.81, P=0.043; A vs. G: OR=1.63, P=0.022). Meanwhile, the expression level of CTLA-4 was remarkably higher in cases compared with the controls. The association analysis indicated that AA genotype carriers exhibited highest level of CTLA-4 (P<0.01). Conclusions: The genotype AA and allele A of CTLA-4 rs231775 polymorphism may have negative effects on HCC by modifying the expression and functions of CTLA-4.
Keywords: Hepatocellular carcinoma, CTLA-4, polymorphism
Introduction
Liver cancer is one of malignant tumors with the highest incidence all over the world [1]. Cause-of-death statistics of Chinese residents reveal that liver cancer is the second leading cause of death among the malignant tumors. The incidence of hepatocellular carcinoma (HCC) in China is the highest worldwide, and HCC brings serious threat to the health of human beings [2]. However, the pathogenesis of HCC has not been well clarified.
Cytotoxic T-lymphocyte antigen-4 (CTLA-4) encoded by CTLA-4 belongs to the immunoglobulin supergene family. Its gene located on chromosome 2 in q33, which is expressed on the surface of T cells in form of dimers [3]. The function of CTLA-4 is mainly to transmit inhibitory signals so as to negatively regulate the proliferation of T cells and block the immune responses [4]. Genetic variations or expression disorders of CTLA-4 may lead to an attenuated inhibitory effect on the activation of T cells, meanwhile autoimmune injury was activated [5,6]. In recent years, studies concerning the relationship between CTLA-4 polymorphisms and some diseases have attracted extensive attention. Previous studies have indicated that CTLA-4 polymorphisms are closely related to various autoimmune diseases such as idiopathic dilated cardiomyopathy (IDCM), lupus erythematosus (LE) and thyroid diseases [7-9]. Just as we have known, CTLA-4 polymorphism exists a mutation from A to G on exon2 as rs231775, the relationship between CTLA-4 rs231775 polymorphism with various diseases has been exported, such as gastric cancer [10], colorectal cancer [11], hepatocellular carcinoma and cervical cancer [12]. But the association of CTLA-4 rs231775 polymorphism with HCC risk is hardly reported.
In present study, a case-control model included 80 cases and 78 controls were conducted. The CTLA-4 rs231775 polymorphism was checked by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. The result about the relevance of CTLA-4 rs231775 polymorphism with HCC risk provides a theoretical basis for the risk prediction and the treatment of HCC.
Subjects and methods
Research subjects
A case-control study was carried out. 80 patients with HCC by pathological examination were set in case group from Jinan Central Hospital, and 78 individuals who had a health examination in same hospital during the same period had no tumors or genetic diseases belonged to the control group. An age gap of ± 2 years existed among individuals in the two groups. Furthermore, the subjects in two groups had the similar native place, gender and ethnicity, as well as parallel life and eating habits, and had no genetic connections. Our research protocol was supported by Research Ethics Committee of the hospital. Written consents were acquired from every subject.
Sample preparation
5 ml peripheral venous blood was collected from each subject with TRANK 303 heparin vacuum blood collection tube. The expression level of CTLA-4 in serum was tested by enzyme linked immunosorbent assay (ELISA) kit.
DNA extraction
Genome DNA of peripheral venous blood from all subjects was extracted by using the conventional phenol-chloroform method. The purity and concentration of DNA were tested by an ultraviolet spectrophotometer and then stored at -20°C refrigerator.
Genotyping
PCR primers were designed by Premier 5.0 according to gene sequence of exon 1 in CTLA-4 gene in Genebank and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Forward primer: 5’-GCTCTACTTCCTGAAGACCT-3’, reverse primer: 5’-AGTCTCACTCACCTTTGCAG-3’. PCR reaction system was a volume of 25 µL solution, which included substrate template DNA (0.5 µg), 1 µL forward primer (0.5 µmol/L), 1 µL reverse primer (0.5 µmol/L), 12.5 µL Master Mix and the corresponding buffer solution. The PCR amplification conditions were as follows: initial denaturation at 95°C for 7 minutes, followed by 35 cycles with 94°C for 45 s, 58°C for 45 s, and 72°C for 1 min and final extension at 72°C for 10 min. The amplification product had a length of 162 bp, and included CTLA-4 rs231775 polymorphism. 10 µL PCR product was digested by Bbv I, and the digested products were checked by 2% polyacrylamide gel electrophoresis (PAGE).
Statistical analysis
The data analysis was performed with SPSS 18.0 software. The genotypes frequencies of the controls were checked whether the distribution was in accordance with Hardy-Weinberg Equilibrium (HWE). Odds ratio (OR) with 95% confidence interval (CI) were used to evaluate the association of CTLA-4 rs231775 polymorphism with the risk of HCC, which was calculated by chi-squared test. In present study, the relationship between CTLA-4 rs231775 polymorphism and the expression of CTLA-4 in serum was analyzed with linear regression analysis. P<0.05 indicates significant difference.
Results
HWE examination
The examination result showed that the genotypes frequencies of CTLA-4 rs231775 polymorphism in control group were satisfied with HWE (P>0.05).
Analysis of CTLA-4 rs231775 polymorphism
The digestion results showed AA genotype with a 162 bp fragment, AG genotype with three fragments of 162 bp, 88 bp and 74 bp, and GG genotype with 88 bp and 74 bp fragments.
Analysis of CTLA-4 level in the serum and CTLA-4 rs231775 polymorphism
The CTLA-4 level in the serum of subjects in the two groups is shown in Table 1. The result revealed that the expression level of CTLA-4 was significantly higher in cases compared with the control group (P<0.05). In the same time, we performed an analysis of the relationship between the expression level of CTLA-4 and genotypes distribution of CTLA-4 rs231775 polymorphism case group. The result showed that in case group, the expression level of CTLA-4 was highest in the AA genotype carriers compared with AG and GG (P<0.01) (Figure 1).
Table 1.
Expression level of CTLA-4 in the serum
| Group | CTLA-4 concentration (µg/L) | P value |
|---|---|---|
| Case group | 1.63 ± 0.76 | 0.022 |
| Control group | 0.49 ± 0.18 |
Figure 1.
The effects of genotype distribution on the level of CTLA-4. **Indicates significant differences.
The relationship of CTLA-4 rs231775 polymorphism with HCC susceptibility
We made a comparison in genotypes and alleles frequencies of CTLA-4 rs231775 polymorphism between the cases and controls. The result demonstrated that frequencies of hemozyous genotype AA and allele A were remarkably higher in case group than controls (18.7% vs. 9.0%, 41.2% vs. 30.1%). We also found that AA genotype and A allele might be risk factors for HCC (AA vs. GG: OR=2.81, 95% CI=1.01-7.78; A vs. G: OR=1.63, 95% CI=1.02-2.59) (Table 2).
Table 2.
The comparison of genotypes and alleles frequencies of CTLA-4 rs231775 polymorphism in case and control group
| CTLA-4 rs231775 | Case group (n=80, %) | Control group (n=78, %) | ORs (95% CIs) | P value |
|---|---|---|---|---|
| GG | 29 (36.3) | 38 (48.7) | 1 (Ref.) | - |
| GA | 36 (45.0) | 33 (42.3) | 1.43 (0.73-2.81) | 0.30 |
| AA | 15 (18.7) | 7 (9.0) | 2.81 (1.01-7.78) | 0.043 |
| G | 94 (58.8) | 109 (69.9) | 1 (Ref.) | - |
| A | 66 (41.2) | 47 (30.1) | 1.63 (1.02-2.59) | 0.039 |
Discussion
The occurrence and development of HCC is a complex biological process influenced by many factors. The environmental factors are the external causes, whereas genetic susceptibility is an important internal cause and largely determines the risk degree of developing liver cancer among individuals [13,14]. Human genes have abundant polymorphisms. Genetic mutations may lead to abnormal expressions of corresponding proteins. As a result, each individual may react differently to toxics or carcinogens, and thus may have different liver cancer susceptibility [15,16]. In recent years, abundant publications have reported genetic variants are related with HCC susceptibility. According to the study of Li et al., genetic variant of CYP2C19 may be an important risk factor for HCC in China Han population, but not in patients with HBV infection [17]. A meta-analysis from Xiao et al. demonstrated the homozyous null genotypes of glutathioneS-transferasegenetic polymorphisms GSTM1 and GSTT1 significantly increased the risk of HCC in Chinese [18].
CTLA-4 is expressed in activated T cells, and is the homologue of CD28 in structure [5]. CTLA-4 can participate as a negative costimulatory molecule in the activation process of T cells and regulate immune responses of the body [19,20]. CTLA-4 polymorphisms can modify the function of CTLA-4, and are related to a variety of autoimmune diseases [21-23]. CTLA-4 rs231775 polymorphism is derived from a Thr to Gly mutations in the signal peptide which may lead to subtle changes in subcellular location of CTLA-4 mature proteins. In the other hand, the interaction between nascent peptide fragments and molecular chaperones was influenced by genetic variants of CTLA-4, which modified the function of CTLA-4 [24,25].
Many reports have shown that CTLA-4 rs231775 polymorphism is correlated with the function of CTLA-4 [26-28]. The G allele of CTLA-4 +49G/A polymorphism is associated with the generation process of CTLA-4 protein by endoplasmic reticulum. The increasing frequency of G allele can reduce the generation and expression of glycosylated proteins in CTLA-4 +49G/A polymorphism. As a result, the regulation of T cells proliferation is weakened, which will damage the inhibitory function of CTLA-4 [29].
In present study, the result showed that the case group had an obviously higher frequencies of genotype AA and allele A than the controls, which implied that AA genotype and A allele of CTLA-4 rs231775 polymorphism might be related to the increasing risk of HCC. In addition, this study also showed that AA genotype had an apparent association with the expression of CTLA-4 in the serum in cases, suggesting that the CTLA-4 rs231775 polymorphism might result in disorders of cellular immune function by affecting secreting of CTLA-4.
In conclusion, a predicted possible mechanism of CTLA-4 rs231775 polymorphism for HCC susceptibility may be as follows: the replacement of Thr to Gly in the signal peptide on CTLA-4 rs231775 polymorphism may change the expression level and function of CTLA-4 and as a result, the polymorphism affects the individuals susceptibility for HCC. However, our study did not consider effects of the environmental factors on the pathogenesis of HCC and the sample size was relatively small, which may influence the reliability of our results. Therefore, further well-design research with large sample size was required to investigate the issue.
Disclosure of conflict of interest
None.
References
- 1.Lu X, Ye K, Zou K, Chen J. Identification of copy number variation-driven genes for liver cancer via bioinformatics analysis. Oncol Rep. 2014;32:1845–1852. doi: 10.3892/or.2014.3425. [DOI] [PubMed] [Google Scholar]
- 2.Chen QW, Cheng CS, Chen H, Ning ZY, Tang SF, Zhang X, Zhu XY, Vargulick S, Shen YH, Hua YQ, Xie J, Shi WD, Gao HF, Xu LT, Feng LY, Lin JH, Chen Z, Liu LM, Ping B, Meng ZQ. Effectiveness and complications of ultrasound guided fine needle aspiration for primary liver cancer in a Chinese population with serum alpha-fetoprotein levels ≤200 ng/ml--a study based on 4,312 patients. PLoS One. 2014;9:e101536. doi: 10.1371/journal.pone.0101536. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Jin Y, Ben S, Teng W, Zhang J, Xiong X, Zhao Y, Huang W. [A linkage study of cytotoxic T lymphocyte-associated antigen 4 gene and Graves’ disease in northern Chinese Han ethnic] . Zhonghua Nei Ke Za Zhi. 2002;41:809–812. [PubMed] [Google Scholar]
- 4.Pankuweit S, Portig I, Maisch B. Pathophysiology of cardiac inflammation: molecular mechanisms. Herz. 2002;27:669–676. doi: 10.1007/s00059-002-2421-4. [DOI] [PubMed] [Google Scholar]
- 5.Soskic B, Qureshi OS, Hou T, Sansom DM. A transendocytosis perspective on the CD28/CTLA-4 pathway. Adv Immunol. 2014;124:95–136. doi: 10.1016/B978-0-12-800147-9.00004-2. [DOI] [PubMed] [Google Scholar]
- 6.Engeland CE, Grossardt C, Veinalde R, Bossow S, Lutz D, Kaufmann JK, Shevchenko I, Umansky V, Nettelbeck DM, Weichert W, Jager D, von Kalle C, Ungerechts G. CTLA-4 and PD-L1 checkpoint blockade enhances oncolytic measles virus therapy. Mol Ther. 2014;22:1949–1959. doi: 10.1038/mt.2014.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Han B, Jiang H, Liu Z, Zhang Y, Zhao L, Lu K, Xi J. CTLA4-Ig relieves inflammation in murine models of coxsackievirus B3-induced myocarditis. Can J Cardiol. 2012;28:239–244. doi: 10.1016/j.cjca.2011.11.014. [DOI] [PubMed] [Google Scholar]
- 8.Sun T, Hu Z, Shen H, Lin D. Genetic polymorphisms in cytotoxic T-lymphocyte antigen 4 and cancer: the dialectical nature of subtle human immune dysregulation. Cancer Res. 2009;69:6011–6014. doi: 10.1158/0008-5472.CAN-09-0176. [DOI] [PubMed] [Google Scholar]
- 9.Gu LQ, Zhu W, Zhao SX, Zhao L, Zhang MJ, Cui B, Song HD, Ning G, Zhao YJ. Clinical associations of the genetic variants of CTLA-4, Tg, TSHR, PTPN22, PTPN12 and FCRL3 in patients with Graves’ disease. Clin Endocrinol (Oxf) 2010;72:248–255. doi: 10.1111/j.1365-2265.2009.03617.x. [DOI] [PubMed] [Google Scholar]
- 10.Kordi-Tamandani DM, Davani SK, Baranzehi T, Hemati S. Analysis of promoter methylation, polymorphism and expression profile of cytotoxic T-lymphocyte-associated antigen-4 in patients with gastric cancer. J Gastrointestin Liver Dis. 2014;23:249–253. doi: 10.15403/jgld.2014.1121.233.dmkt. [DOI] [PubMed] [Google Scholar]
- 11.Solerio E, Tappero G, Iannace L, Matullo G, Ayoubi M, Parziale A, Cicilano M, Sansoe G, Framarin L, Vineis P, Rosina F. CTLA4 gene polymorphism in Italian patients with colorectal adenoma and cancer. Dig Liver Dis. 2005;37:170–175. doi: 10.1016/j.dld.2004.10.009. [DOI] [PubMed] [Google Scholar]
- 12.Hu L, Liu J, Chen X, Zhang Y, Liu L, Zhu J, Chen J, Shen H, Qiang F, Hu Z. CTLA-4 gene polymorphism +49 A/G contributes to genetic susceptibility to two infection-related cancers-hepatocellular carcinoma and cervical cancer. Hum Immunol. 2010;71:888–891. doi: 10.1016/j.humimm.2010.05.023. [DOI] [PubMed] [Google Scholar]
- 13.Ito Y, Miyashiro I, Ito H, Hosono S, Chihara D, Nakata-Yamada K, Nakayama M, Matsuzaka M, Hattori M, Sugiyama H, Oze I, Tanaka R, Nomura E, Nishino Y, Matsuda T, Ioka A, Tsukuma H, Nakayama T. Long-term survival and conditional survival of cancer patients in Japan using population-based cancer registry data. Cancer Sci. 2014;105:1480–1486. doi: 10.1111/cas.12525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Prayong P, Mairiang E, Pairojkul C, Chamgramol Y, Mairiang P, Bhudisawasdi V, Sripa B. An interleukin-6 receptor polymorphism is associated with opisthorchiasis-linked cholangiocarcinoma risk in Thailand. Asian Pac J Cancer Prev. 2014;15:5443–5447. doi: 10.7314/apjcp.2014.15.13.5443. [DOI] [PubMed] [Google Scholar]
- 15.Xu JJ, Zou LY, Yang L, He XL, Sun M. Common polymorphisms in the HIF-1alpha gene confer susceptibility to digestive cancer: a meta-analysis. Genet Mol Res. 2014;13:6228–6238. doi: 10.4238/2014.August.15.5. [DOI] [PubMed] [Google Scholar]
- 16.Tang T, Song X, Yang Z, Huang L, Wang W, Tan H. Association between murine double minute 2 T309G polymorphism and risk of liver cancer. Tumour Biol. 2014;35:11353–11357. doi: 10.1007/s13277-014-2432-9. [DOI] [PubMed] [Google Scholar]
- 17.Li QY, Zhao NM, Wang LC, Duan HF, Ma YC, Zhang W, Zhao HW, Qin YH. Individuals having variant genotypes of cytochrome P450 2C19 are at increased risk of developing primary liver cancer in Han populations, without infection with the hepatitis virus. Tumour Biol. 2014;35:9023–9026. doi: 10.1007/s13277-014-2144-1. [DOI] [PubMed] [Google Scholar]
- 18.Xiao Y, Ma JZ. [Study of the relationship between glutathione S-transferase genetic polymorphisms M1 and T1 and susceptibility to primary liver cancer in Chinese: a meta-analysis] . Zhonghua Gan Zang Bing Za Zhi. 2012;20:774–779. doi: 10.3760/cma.j.issn.1007-3418.2012.10.014. [DOI] [PubMed] [Google Scholar]
- 19.Dariavach P, Mattei MG, Golstein P, Lefranc MP. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains. Eur J Immunol. 1988;18:1901–1905. doi: 10.1002/eji.1830181206. [DOI] [PubMed] [Google Scholar]
- 20.Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, Thompson CB, Griesser H, Mak TW. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science. 1995;270:985–988. doi: 10.1126/science.270.5238.985. [DOI] [PubMed] [Google Scholar]
- 21.Li G, Shi F, Liu J, Li Y. The effect of CTLA-4 A49G polymorphism on rheumatoid arthritis risk: a meta-analysis. Diagn Pathol. 2014;9:157. doi: 10.1186/s13000-014-0157-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Hui L, Lei Z, Peng Z, Ruobing S, Fenghua Z. Polymorphism analysis of CTLA-4 in childhood acute lymphoblastic leukemia. Pak J Pharm Sci. 2014;27:1005–1013. [PubMed] [Google Scholar]
- 23.Han FF, Fan H, Wang ZH, Li GR, Lv YL, Gong LL, Liu H, He Q, Liu LH. Association between co-stimulatory molecule gene polymorphism and acute rejection of allograft. Transpl Immunol. 2014;31:81–86. doi: 10.1016/j.trim.2014.06.003. [DOI] [PubMed] [Google Scholar]
- 24.Gao X, Zhang S, Qiao X, Yao Y, Wang L, Dong D, Ma X, Wang T. Association of cytotoxic T lymphocyte antigen-4 +49A/G polymorphism and cancer risk: An updated meta-analysis. Cancer Biomark. 2014;14:287–294. doi: 10.3233/CBM-140403. [DOI] [PubMed] [Google Scholar]
- 25.Magistrelli G, Jeannin P, Herbault N, Benoit De Coignac A, Gauchat JF, Bonnefoy JY, Delneste Y. A soluble form of CTLA-4 generated by alternative splicing is expressed by nonstimulated human T cells. Eur J Immunol. 1999;29:3596–3602. doi: 10.1002/(SICI)1521-4141(199911)29:11<3596::AID-IMMU3596>3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
- 26.Antczak A, Pastuszak-Lewandoska D, Gorski P, Domanska D, Migdalska-Sek M, Czarnecka K, Nawrot E, Kordiak J, Brzezianska E. Ctla-4 expression and polymorphisms in lung tissue of patients with diagnosed non-small-cell lung cancer. Biomed Res Int. 2013;2013:576486. doi: 10.1155/2013/576486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Lee YH, Song GG. A meta-analysis of the association between CTLA-4 +49 A/G, -318 C/T, and IL-1 polymorphisms and susceptibility to cervical cancer. Neoplasma. 2014;61:481–490. doi: 10.4149/neo_2014_060. [DOI] [PubMed] [Google Scholar]
- 28.Licul V, Cizmarevic NS, Ristic S, Mikolasevic I, Mijandrusic BS. CTLA-4 +49 and TNF-alpha-308 gene polymorphisms in celiac patients with exocrine pancreatic insufficiency. Coll Antropol. 2013;37:1191–1194. [PubMed] [Google Scholar]
- 29.Haller K, Kisand K, Nemvalts V, Laine AP, Ilonen J, Uibo R. Type 1 diabetes is insulin -2221 MspI and CTLA-4 +49 A/G polymorphism dependent. Eur J Clin Invest. 2004;34:543–548. doi: 10.1111/j.1365-2362.2004.01385.x. [DOI] [PubMed] [Google Scholar]