TLR2 and TLR4 polymorphisms influences mRNA and protein expression in colorectal cancer

• Grant number: 15/02342-1
• Support Opportunities: Regular Research Grants - Publications - Scientific article
• Start date: April 01, 2015
• End date: September 30, 2015
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Ana Elizabete Silva
• Grantee: Ana Elizabete Silva
• Host Institution: Instituto de Biociências, Letras e Ciências Exatas (IBILCE). Universidade Estadual Paulista (UNESP). Campus de São José do Rio Preto. São José do Rio Preto , SP, Brazil

Abstract

To evaluate the effect of promoter region polymorphisms TLR2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and also of TLR4+896A/G (rs4986790) on CRC risk. The TLR2-196 to -174del polymorphism was investigated by using allele-specific PCR and the TLR4-1607T/C and TLR4+896A/G by PCR-RFLP (Polymerase Chain Reaction / Restriction Fragment Length Polymorphism). We genotyped 434 DNA samples (194 CRC patients/240 healthy individuals). The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by qPCR 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. 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: OR(95%CI)=1.72(1.03-2.89); P = 0.038 and log-additive: OR(95%CI)=1.59(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 are concordant with the findings regarding mRNA expression. Besides, 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 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). Our study suggests 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. (AU)