|TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer.|
Proença, MA; de Oliveira, JG; Cadamuro, AC; Succi, M; Netinho, JG; Goloni-Bertolo, EM; Pavarino, ÉC; Silva, AE
World journal of gastroenterology
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.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.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 less than 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).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.
|Effect of Helicobacter pylori eradication on TLR2 and TLR4 expression in patients with gastric lesions.|
Cadamuro, AC; Rossi, AF; Matos Biselli-Périco, J; Fucuta Pereira, P; Do Vale, EP; Acayaba, R; Leite, KR; Goloni-Bertollo, EM; Silva, AE
Mediators of inflammation
Helicobacter pylori (Hp) is recognized by TLR4 and TLR2 receptors, which trigger the activation of genes involved in the host immune response. Thus, we evaluated the effect of eradication therapy on TLR2 and TLR4 mRNA and protein expression in H. pylori-infected chronic gastritis patients (CG-Hp+) and 3 months after treatment.A total of 37 patients CG-Hp+ were evaluated. The relative quantification (RQ) of mRNA was assessed by TaqMan assay and protein expression by immunohistochemistry.Before treatment both TLR2 and TLR4 mRNA in CG-Hp+ patients were slightly increased (TLR2 = 1.32; TLR4 = 1.26) in relation to Hp-negative normal gastric mucosa (P ≤ 0.05). After successful eradication therapy no significant change was observed (TLR2 = 1.47; TLR4 = 1.53; P greater than 0.05). In addition, the cagA and vacA bacterial genotypes did not influence the gene expression levels, and we observed a positive correlation between the RQ values of TLR2 and TLR4, both before and after treatment. Immunoexpression of the TLR2 and TLR4 proteins confirmed the gene expression results.In conclusion, the expression of both TLR2 and TLR4 is increased in CG-Hp+ patients regardless of cagA and vacA status and this expression pattern is not significantly changed after eradication of bacteria, at least for the short period of time evaluated.
|Laminin forms an independent network in basement membranes.|
P D Yurchenco,Y S Cheng,H Colognato
The Journal of cell biology
Laminin self-assembles in vitro into a polymer by a reversible, entropy-driven and calcium-facilitated process dependent upon the participation of the short arm globular domains. We now find that this polymer is required for the structural integrity of the collagen-free basement membrane of cultured embryonal carcinoma cells (ECC) and for the supramolecular organization and anchorage of laminin in the collagen-rich basement membrane of the Engelbreth-Holm-Swarm tumor (EHS). First, low temperature and EDTA induced the dissolution of ECC basement membranes and released approximately 80% of total laminin from the EHS basement membrane. Second, laminin elastase fragments (E4 and E1') possessing the short arm globules of the B1, B2, and A chains selectively acted as competitive ligands that dissolved ECC basement membranes and displaced laminin from the EHS basement membrane into solution. The fraction of laminin released increased as a function of ligand concentration, approaching the level of the EDTA-reversible pool. The smaller (approximately 20%) residual pool of EHS laminin, in contrast, could only be effectively displaced by E1' and E4 if the collagenous network was first degraded with bacterial collagenase. The supramolecular architecture of freeze-etched and platinum/carbon replicated reconstituted laminin gel polymer, ECC, and collagenase-treated EHS basement membranes were compared and found to be similar, further supporting the biochemical data. We conclude that laminin forms a network independent of that of type IV collagen in basement membranes. Furthermore, in the EHS basement membrane four-fifths of laminin is anchored strictly through noncovalent bonds between laminin monomers while one-fifth is anchored through a combination of these bonds and laminin-collagen bridges.Full Text Article