Published online Jul 7, 2015. doi: 10.3748/wjg.v21.i25.7730
Peer-review started: October 18, 2014
First decision: December 2, 2014
Revised: January 3, 2015
Accepted: February 12, 2015
Article in press: February 13, 2015
Published online: July 7, 2015
AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor (TLR)2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and of TLR4+896A/G (rs4986790) on colorectal cancer (CRC) risk.
METHODS: The TLR2-196 to -174del polymorphism was investigated using allele-specific polymerase chain reaction (PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length polymorphism (RFLP). We genotyped 434 DNA samples from 194 CRC patients and 240 healthy individuals. The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by quantitative PCR TaqMan assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference genes were used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies.
RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models (log-additive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to -174del was associated with increased CRC risk [dominant: odds ratio (OR) = 1.72, 95%CI: 1.03-2.89; P = 0.038 and log-additive: OR =1.59, 95%CI: 1.02-2.48; P = 0.039]. TLR2 mRNA expression was increased in tumor tissue (RQ = 2.36) when compared to adjacent normal tissue (RQ = 1; P < 0.0001), whereas the TLR4 mRNA showed a basal expression (RQ = 0.74 vs RQ = 1, P = 0.452). Immunohistochemistry analysis of TLR2 and TLR4 protein expression was concordant with the findings of mRNA expression. In addition, the TLR2-196 to -174del variant carriers showed mRNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to -174del variant carriers [117 ± 10 arbitrary unit (a.u.) vs 95 ± 4 a.u., P = 0.03]. However, for the TLR4 -1607T/C polymorphism no significant difference was found for both mRNA (P = 0.56) and protein expression (P = 0.26).
CONCLUSION: Our findings suggest that TLR2-196 to -174del polymorphism increases TLR2 mRNA expression and is associated with higher CRC risk, indicating an important role in CRC genetic susceptibility.
Core tip: This study investigated the influence of the toll-like receptor (TLR)2 and TLR4 functional polymorphisms on mRNA and protein expression levels in colorectal cancer samples and the association of these polymorphisms with the risk of developing this neoplasm. Increased expression of TLR2 (mRNA and protein) in tumor tissue was observed compared with adjacent normal tissue. Moreover, for the first time, the polymorphism TLR2-196 to -174del was associated with a higher risk of developing this type of cancer, and TLR2-196 to -174del allele carriers showed mRNA relative expression approximately two times higher than wild-genotype carriers. Thus, functional polymorphism in TLR2 may change gene expression levels, accentuating inflammation and aggravating the development of colorectal cancer.