Tumor Biol. DOI 10.1007/s13277-014-2902-0

RESEARCH ARTICLE

Toll-like receptor 4 gene polymorphism downregulates gene expression and involves in susceptibility to bladder cancer Yizhen Shen & Meimei Bu & Aimin Zhang & Yi Liu & Baochen Fu

Received: 19 October 2014 / Accepted: 26 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Bladder cancer is the ninth most frequent malignancy in China. Toll-like receptor 4 (TLR4) is expressed on various cells and greatly involves in immune responses. Genetic polymorphism may affect the pathogenesis of diseases through various pathways. In the current study, we evaluated the association between genetic polymorphisms in TLR4 and risk of bladder cancer. We also examined the effect of the polymorphisms on gene expression. The TLR4 −729G/ C and −260G/C polymorphisms were genotyped in 282 bladder cancer patients and 298 healthy controls in the Chinese population. Results showed that subjects with −729GC genotype are at significantly higher risk of bladder cancer than those with GG genotype [odds ratio (OR)=2.50, 95 % confidence interval (CI)=1.39–4.48, P=0.002]. Similarly, TLR4 −729C allele revealed a positive association with the disease (OR = 2.39, P = 0.002). The other polymorphism, TLR4 −260G/C, did not present clear correlations with bladder cancer. To understand the function of the polymorphisms, we evaluated TLR4 messenger RNA (mRNA) and protein levels in CD4+ T cells, CD8+ T cells, and monocytes from subjects carrying different TLR4 genotypes. Results revealed that subjects carrying −729GC genotype had significantly downregulated mRNA and protein levels of TLR4 in CD4+ T cells, CD8+ T cells, and monocytes compared to those carrying GG genotype. However, subjects with −260G/C polymorphism did not show any differences in gene expression from immune cells These data suggest that TLR4 Y. Shen (*) : A. Zhang : Y. Liu : B. Fu Department of Urology, General Hospital of Jinan Military Command, 25 Shifan Road, Jinan, Shandong 250031, People’s of Republic of China e-mail: [email protected] M. Bu The Maternal and Child Health Hospital of Jinan City, Jinan, Shandong 250001, China

polymorphism is associated with increased susceptibility to bladder cancer possibly by downregulating gene expression in various immune cells. Keywords Toll-like receptor 4 . Polymorphism . Bladder cancer

Introduction Bladder cancer belongs to urinary system tumor. It begins most often in the cells that line the inside of the bladder. The cancer typically affects older adults, though it can occur at any age [1]. Smoking and occupational exposure to carcinogens are established risk factors of bladder cancer [2]. Recent studies have shown that dysregulation of the immune system may play critical roles in the development of the disease [3]. Toll-like receptor 4 (TLR4) is a member of the Toll-like receptor (TLR) family, which plays a fundamental role in pathogen recognition and activation of innate immunity [4]. This protein can be widely detected on various cells and tissues, such as CD4+ T cells, CD8+ T cells, monocytes, adipose tissue, skeletal muscle, etc. [5]. The protein is originally known as a defense against microbial infection [6, 7]. TLR4 activates two distinct pathways originating from different cellular locations, the cell surface, and the endosome. This results either in inflammatory responses mediated by the adaptor MyD88 or antiviral signaling responses transduced by TRAM/TRIF and IRF3 [8, 9]. TLR4 signaling also affects immune responses in tumors. Studies have shown that dysregulation of TLR4 causes a decrease of CD8+ T cell cytotoxicity. Moreover, TLR4 pathway may control DC maturation and antigen presentation [10, 11]. All the results suggest a protective role of the protein in the oncogenesis. On the other hand, stimulation of TLR4 in regulatory T cells may cause cell proliferation and suppresses antitumor activity [12, 13]. In

Tumor Biol.

addition, many tumor cells and tumor tissues have been found to express TLRs [14], indicating that TLR4 may affect the development of cancer through various pathways. Genetic single-nucleotide polymorphisms (SNPs) may affect the development of human diseases. Two SNPs in TLR4 gene are largely studied. The +896A/G polymorphism causes an amino acid exchange (aspartate to glycine) at position 299 (Asp299Gly), and the +1196C/T polymorphism causes a switch from threonine to isoleucine at position 399 (Thr399Ile) [15]. Researches have revealed that the two polymorphisms are associated with different diseases such as gastric cancer, pyelonephritis, hepatitis C virus-induced hepatocellular carcinoma, etc. [16–18]. Previously, we have reported that the TLR4 +3725G/C polymorphism is associated with an increased risk of bladder cancer [19]. Here, we detected two novel TLR4 polymorphisms (rs11536865 −729G/C and rs41391946 −260G/C) and investigated their associations with bladder cancer in the Chinese population. We also examined whether these two polymorphisms could affect gene expression in different immune cells.

Materials and methods Study subjects The study population consisted of 282 patients. All cases were diagnosed with bladder transitional cell carcinoma and confirmed by histological examinations. Patients with a family history of cancers, autoimmune diseases, and other cancers were excluded from the research. The control group included 298 healthy subjects who came to the hospital for general health exams. The controls were genetically unrelated cancerfree individuals living in the same residential areas and frequency matched to the cases on age, sex, smoking status, and alcohol use. Definition of smoking in the study was individuals who smoked 20 cigarettes a day for over 10 years. The study was approved by the Review Board of the General Hospital of Jinan Military Command. Written informed consent was obtained from each participant.

for 30 s, and 72 °C for 30 s, with final elongation at 72 °C for 5 min on the GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA, USA). PCR products were then digested with the restriction enzymes Taq I and Pst I (New England Biolabs, Beverly, MA, USA), respectively. The digested PCR products were fractionated on 2 % agarose Tris–borate–EDTA gel (Agarose 1000; Gibco BRL, Rockville, MD, USA) and stained with ethidium bromide. To confirm the genotyping results, more than 10 % of PCRamplified DNA samples were examined by DNA sequencing. Results between PCR and DNA sequencing analysis were 100 % concordant. Real-time quantitative reverse transcriptase and Western blot Real-time quantitative reverse transcriptase PCR (RT-PCR) was performed using the Exicycler 96 real-time RT-PCR system (A-2060; Bioneer, Daejeon, South Korea) and SYBR Green Master Mix (SY1020; Solarbio, Beijing, China). Amplification was performed under the following conditions: 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Experiments were performed in triplicate in the same reaction. The results of the real-time quantitative RT-PCR experiments were calculated using the 2(−Delta Delta C(T)) method. Western blot was conducted according to a previously published method. Statistical analysis The SPSS statistical software package ver. 13.0 (SPSS Inc., Chicago, IL, USA) and the Prism 5.0 were used for statistical analysis. Demographic data between the study groups were compared using the chi-square test and the Student’s t test. Hardy–Weinberg equilibrium was analyzed using the chisquare test. For SNP analysis, genotype and allele frequencies of TLR4 were compared between groups using the chi-square test and odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated using unconditional logistic regression. Student’s t test was used to compare messenger RNA (mRNA) and protein levels between different genotypes. P values less than 0.05 were considered significant.

DNA extraction and genotyping DNA extraction was conducted using previously published methods [19]. The TLR4 −729G/C and −260G/C polymorphisms were detected by the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. In brief, PCR was performed in a 20-μL mixture containing 200 ng of genomic DNA, 1.5 mM MgCl2, 0.5 μM of each primer, 2 μL of 10× PCR buffer, 0.2 mM dNTP, and 1.2 U Taq polymerase (all purchased from Qiagen Inc., Hilden, Germany). After an initial denaturation at 95 °C for 5 min, the DNA was amplified for 30 cycles at 95 °C for 30 s, 55 °C

Results Characteristics of the study population Selected characteristics of the cases and controls are presented in Table 1. Bladder cancer cases and controls did not show a significant difference in regard to age, sex, body mass index (BMI), or hematological parameters. As expected, there were more ever smokers in the cases than in the controls (Table 1).

Tumor Biol. Table 1

Distribution of selected demographic variables and risk factors among the cancer cases and healthy controls

Characteristic

Cancer patients (n=282)

Controls (n=298)

P value

Age (years) Sex no. (%) Male Female BMI (kg/m2) Hematological parameters Hemoglobin (g/L) White blood cells (109/L) Red blood cells (1012/L) Smoking status Never Ever Histopathological grade G1/G2 G3

62.8±12.5

62.5±11.3

>0.05 >0.05

185 (65.6) 97 (34.4) 23.5±7.8

202 (67.9) 96 (32.1) 23.2±10.6

136±42 5.63±2.38 4.79±0.56

135±47 5.55±2.17 4.81±0.78

143 (32.8) 293 (67.2)

314 (60.2) 208 (39.8)

>0.05 >0.05 >0.05 0.05). As for the TLR4 −729G/C SNP, the prevalence of GC Table 2

>0.05

genotype was significantly higher in patients than in controls (13.8 versus 6.0 %; OR=2.50, 95 % CI=1.39–4.48, P= 0.002). The −729C allele also revealed significantly increased frequency in bladder cancer patients compared to healthy donors (OR=2.39, 95 % CI=1.39–4.22, P=0.002). As for the TLR4 −260G/C SNP, the prevalence of GG genotype and GC genotype was 92.9 and 7.1 % in patients and 93.6

The distribution of TLR4 SNPs in patients and controls

Polymorphisms

Cases (N=282) (%)

Controls (N=298) (%)

OR (95 % CI)

P value

243 (86.2) 39 (13.8)

280 (94.0) 18 (6.0)

Ref. 2.50 (1.39–4.48)

0.002*

525 (93.1) 39 (6.9)

578 (97.0) 18 (3.0)

Ref. 2.39 (1.39–4.22)

0.002*

262 (92.9) 20 (7.1)

279 (93.6) 19 (6.4)

Ref. 1.12 (0.59–2.15)

0.731

544 (96.5) 20 (3.5)

577 (96.8) 19 (3.2)

Ref. 1.12 (0.59–2.12)

0.736

507 (89.9) 18 (3.2) 37 (6.6) 2 (0.3)

560 (94.0) 18 (3.0) 17 (2.9) 1 (0.1)

Ref. 1.11 (0.57–2.15) 2.40 (1.34–4.32) 2.21 (0.20–24.45)

0.769 0.003* 0.507

TLR4 −729G/C Genotype GG GC Allele G C TLR4 −260G/C Genotype GG GC Allele G C Haplotypes (−729, −260) GG GC CG CC *P

Toll-like receptor 4 gene polymorphism downregulates gene expression and involves in susceptibility to bladder cancer.

Bladder cancer is the ninth most frequent malignancy in China. Toll-like receptor 4 (TLR4) is expressed on various cells and greatly involves in immun...
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