The Effects Of Toll-like Receptor 2 On Bioactivity Of Human Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common digestive system malignancies in our country, with high morbidity and mortality. Up till now, pathogenesis of hepatocellular carcinoma is not completely clear, and there is lacking of effective treatment. So, to study the pathogenesis of hepatocellular carcinoma and search for new therapeutic target and effective treatment is still an urgent task. Toll-like receptor 2 (TLR2) is key innate immunity receptor participating in nonspecific immune response. TLR2 signaling is regarded as one of the mechanisms of chronic inflammation, but it can also mediate tumor cell immune evasion and tumor development. However, the role of TLR2 in the progression of HCC remains to be further studied.ObjectiveTo examine the effects and mechanism of TLR2 on the bioactivity of HCC cell lines, HepG2 and BEL-7402.Method1 Two cell lines (HepG2 and BEL-7402) were used in our experiment. The expression of TLR2 in HepG2 and BEL-7402 was assayed by Western blot.2 Transfection of siRNA:Three specific small interfering RNAs of TLR2 (TLR2-siRNAs) were synthesized. HepG2 and BEL-7402 cells were transfected with TLR2-siRNAs. Real Time PCR and Western blot tests were used to detect the interference efficiency of TLR2-siRNAs, and screen the siRNA with the highest interference efficiency for subsequent experimental treatment. Experimental groups: Three groups were blank group, scramble group and TLR2-siRNA group.3 recombinant high mobility group boxl (rHMGB1) treatment:TLR2-siRNA transfected cells were treated with rHMGB1. Four groups were blank group, rHMGB1 group, scramble+rHMGB1 group and TLR2-siRNA+ rHMGB1 group.4 Detection:Using MTT assay, transwell and flow cytometry, we detected the changes of proliferation, migration, invasion and apoptosis of human hepatocellular carcinoma cells.5 The expression of NF-κB/P65 was assayed by Western blot.Results1 Results of Western blot showed that TLR2 was expressed in HepG2 and BEL-7402 cells.2 TLR2-siRNA obviously inhibited the expression of TLR2.The results of real time PCR and western blot demonstrated that the expression of TLR2 mRNA and protein in the siRNA groups was markedly lower than that in the blank groups and scramble groups (P<0.001), and TLR2-siRNA2 has the highest interference efficiency.3 rHMGB1 enhanced the ability of proliferation, invasion, and migration, and inhibited cells apoptosis. However, TLR2-siRNA resulted in a significant inhibition of proliferation, invasion, migration and the promoting effect of rHMGB1, and elevated apoptotic ratio.MTT results showed that the proliferation of TLR2-siRNA group significantly decreased compared with scramble group and blank group (P<0.001), and the proliferation capacity of rHMGB1 group significantly increased in contrast with blank group and rHMGB1+TLR2-siRNA group (P<0.001). Transwell test showed that the migration and invasion ability of TLR2-siRNA group significantly declined compared with the scramble group and blank group (P<0.01), and the migration and invasion ability of rHMGB1 group significantly increased in contrast with blank group and rHMGBl+TLR2-siRNA group (P<0.001). Flow cytometry results showed that the apoptosis ratio of TLR2-siRNA group was significantly higher than that of the scramble group and blank group (P<0.001), and the apoptosis ratio of rHMGB1 obviously decreased compared with rHMGB1+TLR2-siRNA group and blank group.4 rHMGB1 increased the expression of NF-κB/P65 protein, but TLR2-siRNA inhibited the expression of NF-κB/P65 protein and the effect of rHMGB1 on the expression of NF-κB/P65 protein.Western blot showed that the expression of NF-κB/P65 in TLR2-siRNA group significantly decreased compared with the scramble group and blank group (P<0.001), and the expression of NF-κB/P65 in rHMGB1 group obviously increased compared with rHMGB1+TLR2-siRNA group and blank group (P<0.01).Conclusions1 TLR2 and high mobility group boxl (HMGB1) could promote HCC cell proliferation, migration, invasion, and inhibit apoptosis. The downregulation of TLR2 obviously reduced the effect of rHMGB1. So, TLR2 may be one of downstream signaling molecules mediating the tumor-promoting effect of HMGB1.2 HMGB1-TLR2 signaling pathway promotes the development of HCC via NF-KB/P65.3 TLR2 is expected to become a new therapeutic target of HCC.