| Objective: To investigate the expression and role on invasion and metastasis associated with TLR2 in human gastric cancer SGC-7901 cells, and we searched the expression of TLR2 and their clinical significance in gastric cancer tissues to futher study the molecular mechanisem of TLR2 mediating gastric cancer metastasis.Methods: (1)The experiments were divided into three groups: the control group (SGC-7901 cells without any treatment),the stimulated group (SGC-7901 cells stimulated with LPS), the interrupted group (SGC-7901 cells treated with LPS and anti-TLR2Ab ); (2)TLR1- TLR10 mRNA expressions of the control were detected by RT-PCR; (3)TLR2, VEGF and MMP-9 mRNA expressions of three groups cells were detected by RT-PCR and Real-time PCR; (4) TLR2 protein expression was detected by flow cytometry (FCM) and Western-blotting; (5)Transwell experiments were carried out to observe the changes in cell invasion and metastasis capacity of three groups; (6)The supernatant IL-6, IL-8,VEGF,MMP-9 of three groups cells were detected by ELISA.(7) TLR2 expression of in 47 gastric cancer samples and 25 normal gastric samples were detected by Ultrasensitive Streptavidin- Peroxidase immunohistochemistry staining to analyze the relationship between the TLR2 expression and the clinical pathological data.Results:(1)Human gastric cancer cells SGC-7901 expressed TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 at mRNA levels. TLR2 was found to be lowly expressed in SGC-7901; (2) The mRNA expression of TLR2, VEGF, MMP-9 of the stimulated group was obviously higher than that of the control group respectively ,and the expression in the stimulated group was 41.50±2.21,8.60±0.93,7.22±0.51 times as much as that in the control group respectively (P<0.01). The expression in the interrupted group was significantly lower than that in the stimulated group, and the expression in the interrupted group was 0.14±0.03, 0.31±0.02, 0.22±0.01 times as much as that in the stimulated group respectively (P<0.01). (3) FCM results showed that the protein expression of TLR2 of the stimulated group was obviously higher than that of the control group, the protein expression of TLR2 by FCM in the stimulated group was 14.50±0.82 times as much as that of the control group, the interrupted group was significantly lower than that of the stimulated group, and the interrupted group was 0.16±0.04 times as much as that of the stimulated group (P<0.01);The result of Western-blotting corresponded to those of FCM.(4) Transwell experiments were carried out to observe the invasion ability of the three groups. The results showed that the invasion cell number was 638±9/each visual field in the stimulated group, which was obviously higher than that of the control group with 125±5/each visual field (P<0.01); However, there was 128±6/each viual field in the interrupted group, which was much lower than that of the stimulated group (P<0.01). (5) ELISA showed that the level of IL-6 in the stimulated group increased significantly as compared with controls,the interrupted group decreased significantly as compared with the stimulated group(P<0.05).And these were no significant differences of IL-8,VEGF,MMP-9 in the three groups cell (P>0.05) .(6)The positive expression rate of TLR2 in gastric cancer tissues and normal gastric tissues was 68.1% and 16.0% respectively. The expression rate of TLR2 was higher in gastric cancer tissues than that of normal gastric tissues(P<0.001).The relationship between positive expression rate of TLR2 and lymph node metastasis, TNM stage and distant metastasis was significant(P<0.05) .However, these was no significant correlation between expression of TLR2 in gastric caner and age,sex, differential degree. (P>0.05). Conclusions: (1) These studies demonstrated that human gastric cancer cell lines SGC-7901 expressed multiple TLRs, including TLR1-TLR9. TLR2 played a role in the process of invasion and metastasis in human gastric cancer SGC-7901 cells. Interruption of TLR2 could effectively suppress the invasion capacity of human gastric cancer SGC-7901 cells. This may provide a new therapy target against gastric cancer metastasis. (2) Combined with clinical data, TLR2 may participate in the occurrence, progression, lymph node metastasis, TNM stages and distant metastasis of gastric cancer.This may provide an important theory evidence for occurrence, metastasis, and therapy of gastric cancer. |
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