Biomedical Research

Abstract

Purpose: Toll-like receptors (TLR) act as a vital innate immunity receptors involved in immune response. More and more evidence implies that mutations of TLR2 gene are correlated with cancers. This retrospective study aimed to identify variants in TLR2 affecting HCC (Hepatocellular Carcinoma) susceptibility.

Methods: Four tgSNPs (tag SNPs) were chose from HapMap data and genotyped in 274 patients with HCC and 277 healthy controls.

Results: Rs3804099 genotype CT (P=0.0001, OR=2.00) and allele T (P=0.019, OR=1.37) showed significant difference between HCC and HC (Healthy Control). People who carried rs7656411 genotype TT (P=0.002, OR=2.18) and allele T (P=0.002, OR=1.46) had a significant association with increased HCC risk. The results were consistent with previous results in dominant or recessive model (P=0.001, OR=1.81; P=0.014, OR=1.58; P=0.009, OR=1.78).

Conclusions: Rs3804099 polymorphism C/T and rs7656411 polymorphism G/T in TLR2 were significantly correlated with HCC susceptibility.

Keywords

Toll-like receptor 2 (TLR2), Genetic polymorphism, Hepatocellular carcinoma.

Introduction

Liver cancer is one of the most frequent cancer or the most frequent cause of cancer-related death around the world [1]. About 70% to 85% of the liver cancer is comprised of Hepatocellular Carcinoma (HCC) [1]. East or Southeast Asia and sub-Saharan Africa have the highest HCC incidence rates. The rate varies greatly between males and females. Chronic hepatitis (such as hepatitis B virus (HBV) or hepatitis C virus (HCV) infection), alcohol drinking, aflatoxin B1 exposure, diabetes, cigarette smoking, and some genetic factors are risks for HCC [2]. In China, about 94 million individuals are hepatitis B surface antigen (HBsAg) positive, which result in a high HCC rate [1,3].

Toll-like receptors (TLRs) is one of transmembrane receptors that act an important role in immune response against microbial pathogens [4]. TLR signals are involved in many liver diseases (hepatitis B or hepatitis C, alcoholic liver disease, primary biliary cirrhosis, non-alcoholic liver diseases, hepatic fibrosis, etc.) [5]. In the liver, various cell types (such as Kupffer cells, hepatocytes and biliary epithelium) can express TLR2 [5]. In HBsAg seropositive patients, expression of TLR2 is downregulated in peripheral blood monocytes, Kupffer cells and hepatocytes [6]. However, in HBsAgseronegative patients, expression of tumor necrosis factor-a and TLR2 is up-regulated [6]. In hepatoma cell lines, stimulating IL-1 receptor or TLR2 receptors could inhibit HBV replication and result in a signaling cascade [7]. Activation of TLR2 was correlated with tumorigenesis in liver in a drugprimed mouse model [8]. Accumulating evidences implying that TLR2 genetic polymorphisms are associated with risk of multiple cancers such as gallbladder cancer, cervical cancer, non-Hodgkin lymphoma, and endometrial cancer [9-12].

We hypothesized that SNPs of TLR2 may be indeed associated with susceptibility to HCC. In order to verify the hypothesis, we conducted this study to characterize the association between TLR2 polymorphisms and HCC in Chinese han population.

Materials and Methods

Study populations

In this retrospective study, we got blood samples from unrelated Chinese han persons in our hospital, Chongqing, China between February 2012 and June 2015. A total of 551 individuals including 274 patients with HCC and 277 healthy controls (HC) were studied. HCC was diagnosed by clinical and biological criteria, and confirmed by examination of iconography (computerized tomography and/or magnetic resonance imaging). Clinical classification was conducted by the tumor-node-metastasis staging system (TNM) from International Union Against Cancer (UICC). Child-Pugh score was used to evaluate the severity of liver disease between HCC and HC groups.

We defined HBV carriers as positive for both HBsAg and HBcAb. HCC patients could have the following treatments: transcatheter arterial chemoembolization, chemotherapy, hepatectomy and liver transplantation. During the period of this study, we randomly selected healthy controls who attending hepatitis examination to eliminate the confounding factor of HBV infection. The criteria for healthy controls were no history of any cancers, and its frequency must match to the HCC patients on gender and age. The study protocol was approved by the ethics committee of our hospital. Written informed consents were obtained from all subjects.

Tagging SNP selection

We chose 4 tgSNPs from TLR2 gene on the International HapMap Project, release27 (http://hapmap.ncbi.nlm.nih.gov) in the CHB population. The exclution criterion: an r2 value<0.8, MAF<0.05. SNPs that seemed to have a function in the literature or are non-synonymous SNP in NCBI were selected firstly.

DNA extraction and SNP analysis

According to manufacturer’s instructions of the Wizard® Genomic DNA Purification Kit (Promega, USA), genomic DNA samples were extracted from peripheral blood leukocytes. We used a NanoDrop spectrophotometer to measure DNA samples, diluted to 30 ng/μl, and stored at -80°C for genotyping. We genotyped the five SNPs with IplexGOLD chemistry on SEQUENOM mass spectrometer (SequenomInc, San Diego, USA). A negative control and duplicate samples were set in every 96-well plate for quality control.

Statistical analysis

We evaluated the risk of HCC patients according to genotypes and alleles of TLR2 in comparison to the HC groups. We used HWE software to test Hardy-Weinberg equilibrium in both groups. Differences in gender, age, and Clinical Characteristics were compared between groups with χ² or Mann-Whitney U test. Each genotype was estimated with dominant or recessive genetic models. Binary logistic regression was used for assessing the relative risks of SNPs. Haploview software (v4.2) was used to identified haplotype frequencies. We carried out SPSS 21.0 (SPSS Inc., Chicago, IL, USA) for statistical calculations and P<0.05 were thought to indicate satistical significance.

Results

In HC group, all the four genotyped SNPs of TLR2 were in accordance with the Hardy-Weinberg equilibrium.

Characteristics of the study population

Demographic and clinical characteristics of the study population are given in Table 1. There were no significant differences in gender, age, HBV carriers and Child-Pugh score between HCC and HC groups (P>0.05).

Table 1. Demographic and clinical characteristics of the study population.

Correlations of SNPs with HCC risks

We studied the correlation of the four SNPs in TLR2 between HCC and HC groups. Rs3804099 genotype CT (P=0.0001, OR=2.00) and allele T (P=0.019, OR=1.17) showed significant difference between HCC and HC. People who carried rs7656411 genotype TT (P=0.002, OR=2.18) and allele T (P=0.002, OR=1.21) had a significant association with increased HCC risk (Table 2). In dominant or recessive model, the results were consistent with previous results (P=0.001, OR=1.81; P=0.014, OR=1.26; P=0.009, OR=1.33). The other two SNPs (rs7696323 and rs11938228) showed no significant correlation with HCC risk (Table 3).

Table 2. Association of TLR2 (rs3804099 and rs7656411) in HCC VS HC.

Table 3. Association of TLR2 (rs11938228 and rs7696323) in HCC VS HC.

Finally, we did a haplotype analysis. Rs7696323 and rs11938228 were in high LD. But none of the haplotypes were significantly associated with HCC risk.

Discussion

In this study, we chose four SNPs in TLR2 to evaluate the risk of HCC in Chinese Han population. We found evidence of variants in TLR2 (rs3804099 and rs7656411) were on the risk of HCC. Toll-like receptors (TLRs) is one of transmembrane receptors that act an important role in immune response against microbial pathogens [4]. TLR2 is proved to be associated with multiple cancers including CRC [13-16]. Researchers have explored the association between TLR2 variants and tumors. Microsatellite GT polymorphism in TLR2 was reported to be associated with sporadic colorectal cancer in Croatian [17]. Carriers with TLR2 (Delta22) polymorphisms were correlated with an increased risk for gallbladder cancer [18]. Another study showed significantly correlation between TLR2 (-196 to -174 del) and cervical cancer susceptibility [10]. Subsequently, the frequency of the TLR2 -196 to -174 del allele was found to be significantly higher in patients with HCV-correlated HCC than those without HCC [19]. Stimulation of monocytes in carriers with the TLR2 -196 to -174 del allele had significantly lower TLR2 and IL-8 expression levels than in those with the TLR2 -196 to -174 ins/ins. The current study found that TLR2 polymorphisms influence the risk of HCC. In our study, rs3804099 genotype CT (P=0.0001, OR=2.00) and allele T (P=0.019, OR=1.17) showed significant difference between HCC and HC. People who carried rs7656411 genotype TT (P=0.002, OR=2.18) and allele T (P=0.002, OR=1.21) had a significant association with increased HCC risk. In dominant or recessive model, the results were consistent with previous results (P=0.001, OR=1.81; P=0.014, OR=1.26; P=0.009, OR=1.33). The other two SNPs (rs7696323 and rs11938228) showed no significant correlation with HCC risk. Our data implied that variations in TLR2 may act an important risk role in HCC risk.

TLR2 rs3804099 is a synonymous SNP. Synonymous SNPs have been assumed inconsequential for a long time, as they do not result in change of polypeptide structure. However, this concept change as synonymous mutations is confirmed to be implicated in diseases in many studies over the last decade. It effect on gene function may through the following mechanisms: (1) Perturbations of mRNA splicing [20,21]; (2) The stability of mRNA [22]; (3) mRNA structure [23]; (4) Protein folding [24,25]. Rs7656411 located in in 3’-UTR of TLR2 with a change from G to T. Rs7656411 was found to be associated with colon cancer [26], Asthma [27]. SNP in 3’- UTR may act its function with miRNA or alone by influence of mRNA splicing or the stability of mRNA, and influence the expression level of TLR2. We found that both the rs3804099 and rs7656411 were significantly correlated with HCC susceptibility and might influence the expression level of TLR2. However, the exact mechanisms of might be different as they located in different regions of TLR2. In collusion, we found rs3804099 polymorphism C/T and rs7656411 polymorphism G/T in TLR2 were significantly associated with HCC susceptibility. Our study has several limitations: lack of a validation assay and a relatively small sample size. In the future, we need a prospective longitudinal study to confirm the findings in larger populations.

References

1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: 69-90.
2. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557-2576.
3. Yin J, Zhang H, He Y, Xie J, Liu S, Chang W. Distribution and hepatocellular carcinoma-related viral properties of hepatitis B virus genotypes in Mainland China: a community-based study. Cancer Epidem Biomar 2010; 19: 777-786.
4. Andreakos E, Foxwell B, Feldmann M. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation? Immunol Rev 2004; 202: 250-265.
5. Testro AG, Visvanathan K. Toll-like receptors and their role in gastrointestinal disease. J Gastroenterol Hepatol 2009; 24: 943-954.
6. French SW, Oliva J, French BA, Li J, Bardag-Gorce F. Alcohol, nutrition and liver cancer: role of Toll-like receptor signaling. World J Gastroenterol 2010; 16: 1344-1348.
7. Thompson AJ, Colledge D, Rodgers S, Wilson R, Revill P, Desmond P. Stimulation of the interleukin-1 receptor and Toll-like receptor 2 inhibits hepatitis B virus replication in hepatoma cell lines in vitro. Antivir Ther 2009; 14: 797-808.
8. Oliva J, Bardag-Gorce F, French BA, Li J, McPhaul L, Amidi F. Fat10 is an epigenetic marker for liver preneoplasia in a drug-primed mouse model of tumorigenesis. Exp Mol Pathol 2008; 84: 102-112.
9. Ashton KA, Proietto A, Otton G, Symonds I, McEvoy M, Attia J. Toll-like receptor (TLR) and nucleosome-binding oligomerization domain (NOD) gene polymorphisms and endometrial cancer risk. BMC Cancer 2010; 10: 382.
10. Pandey S, Mittal RD, Srivastava M, Srivastava K, Singh S, Srivastava S. Impact of Toll-like receptors [TLR] 2 (-196 to -174 del) and TLR 4 (Asp299Gly, Thr399Ile) in cervical cancer susceptibility in North Indian women. Gynecol Oncol 2009; 114: 501-505.
11. Purdue MP, Lan Q, Wang SS, Kricker A, Menashe I, Zheng TZ. A pooled investigation of Toll-like receptor gene variants and risk of non-Hodgkin lymphoma. Carcinogenesis 2009; 30: 275-281.
12. Srivastava K, Srivastava A, Kumar A, Mittal B. Gallbladder cancer predisposition: a multigenic approach to DNA-repair, apoptotic and inflammatory pathway genes. PLoS One 2011;6: e16449.
13. Castano-Rodriguez N, Kaakoush NO, Pardo AL, Goh KL, Fock KM, Mitchell HM. Genetic polymorphisms in the Toll-like receptor signalling pathway in Helicobacter pylori infection and related gastric cancer. Hum Immunol 2014; 75: 808-815.
14. Devi KR, Chenkual S, Majumdar G, Ahmed J, Kaur T, Zonunmawia JC. TLR222 (-196-174) significantly increases the risk of breast cancer in females carrying proline allele at codon 72 of TP53 gene: a case-control study from four ethnic groups of North Eastern region of India. Tumour Biol 2015; 36: 9995-10002.
15. Lu CC, Kuo HC, Wang FS, Jou MH, Lee KC, Chuang JH. Upregulation of TLRs and IL-6 as a marker in human colorectal cancer. Int J Mol Sci 2015; 16: 159-177.
16. Xin C, Zhang H, Liu Z. MiR-154 suppresses colorectal cancer cell growth and motility by targeting TLR2. Mol Cell Biochem 2014; 387: 271-277.
17. Boraska Jelavic T, Barisic M, Drmic Hofman I, Boraska V, Vrdoljak E, Peruzović M. Microsatelite GT polymorphism in the toll-like receptor 2 is associated with colorectal cancer. Clin Genet 2006; 70: 156-160.
18. Srivastava K, Srivastava A, Kumar A, Mittal B. Significant association between toll-like receptor gene polymorphisms and gallbladder cancer. Liver Int 2010; 30: 1067-1072.
19. Nischalke HD, Coenen M, Berger C, Aldenhoff K, Muller T, Berg T. The toll-like receptor 2 (TLR2) -196 to -174 del/ins polymorphism affects viral loads and susceptibility to hepatocellular carcinoma in chronic hepatitis C. Int J Cancer 2012; 130: 1470-1475.
20. Fedetz M, Matesanz F, Caro-Maldonado A, Fernandez O, Tamayo JA, Guerrero M. OAS1 gene haplotype confers susceptibility to multiple sclerosis. Tissue Antigens 2006; 68: 446-449.
21. Solis-Anez E, Delgado-Luengo W, Borjas-Fuentes L, Zabala W, Arraiz N, Pineda L. Molecular analysis of the GABRB3 gene in autistic patients: an exploratory study. Invest Clin 2007; 48: 225-242.
22. Hunt R, Sauna ZE, Ambudkar SV, Gottesman MM, Kimchi-Sarfaty C. Silent (synonymous) SNPs: should we care about them? Methods Mol Biol 2009; 578: 23-39.
23. Shen LX, Basilion JP, Stanton VP Jr. Single-nucleotide polymorphisms can cause different structural folds of mRNA. Proc Natl Acad Sci USA 1999; 96: 7871-7876.
24. Chamary JV, Parmley JL, Hurst LD. Hearing silence: non-neutral evolution at synonymous sites in mammals. Nat Rev Genet 2006; 7: 98-108.
25. Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 2002; 3: 285-298.
26. Slattery ML, Herrick JS, Bondurant KL, Wolff RK. Toll-like receptor genes and their association with colon and rectal cancer development and prognosis. Int J Cancer 2012; 130: 2974-2980.
27. Qian FH, Zhang Q, Zhou LF, Jin GF, Bai JL, Yin KS. Polymorphisms in the toll-like receptor 2 subfamily and risk of asthma: a case-control analysis in a Chinese population. J Investig Allergol Clin Immunol 2010; 20: 340-346.