TLR2 and TLR4 polymorphisms, gene and protein expression in risk of sporadic colorectal cancer


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Marcela Alcântara Proença1, Aline Cristina Targa Cadamuro1, Juliana Garcia Oliveira5, Patrícia Matos Biselli Chicote2, Joice Matos Biselli1, Katia Ramos Moreira Leite4, Geni Satomi Cunrath3, João Gomes Netinho3, Érika Cristina Pavarino2, Eny Maria Goloni Bertollo2, Ana Elizabete Silva1
1UNESP – São Paulo State University, São José do Rio Preto, Brazil, 2FAMERP – São José do Rio Preto Medical School, São José do Rio Preto, Brazil, 3Base Hospital, São José do Rio Preto, Brazil, 4Sírio Libanês Hospital, São José do Rio Preto, Brazil, 5USC - Universidade do Sagrado Coração, Bauru, Brazil

Background

Colorectal cancer (CRC) is one of the inflammation-cancer association models. Polymorphisms in genes involved in the inflammatory process may be interesting targets for potential CRC molecular markers. We aimed to evaluate the association of functional polymorphisms TLR2-196to-174del, TLR4-1607T/C and TLR4+896A/G and risk factors (gender, age, smoking and drinking) with CRC development, and their influence on relative expression levels of mRNA and proteins.

Method

Altogether 434 DNA samples (194 patients/240 controls) were genotyped from peripheral blood leukocytes or tumor tissue cells by PCR/RFLP. Multiple logistic regression was performed to evaluate the association between the polymorphisms and CRC risk (log-additive, dominant and recessive models), adjusted to risk factors. Relative quantification (RQ) of mRNA and protein expression were performed in 40 and 19 tumor samples, by qPCR and immunohistochemistry, respectively.

Results

The variant TLR2-196to-174del was associated with increased risk of CRC (dominant: OR=1.72, 95%CI=1.03 to 2.89, p=0.038 and log-additive: OR=1.59, 95%CI=1.02 to 2.48, p=0.039), but for TLR4-1607T/C and TLR4+896A/G no association was found. Age above 60 (OR=1.83, 95%CI=1.26 to 2.90, p=0.003) and alcohol consumption (OR=2.78, 95%CI=1.68 to 4.60, p=0.000) are also associated with increased risk for CRC. RQ of mRNA showed a significant increase in TLR2 expression (RQ=2.36) in tumor tissue when compared with adjacent normal tissue (p<0.0001), while for TLR4 no difference was observed (RQ=0,74). In agreement, the immunohistochemistry showed positive immunostaining in 84,2% of tumors samples for TLR2 and 50% for TLR4. When tumor samples were stratified according to the polymorphisms in promoter region, carriers of TLR2-196to-174del variant had a median RQ for mRNA about 2-fold higher (RQ=2.21) when compared with wild genotype (p=0.035). However, there was no influence of TLR4-1607T/C on gene expression.

Conclusion

The variantTLR2-196to-174del is associated with increased risk of CRC in Brazilian population, and seems to increase TLR2 mRNA expression in tumor tissue, suggesting an important role in CRC.