TLR4 Activation Promotes The Invasion Ability Of Hepatic Cancer Cells

Backgroud:Hepatic cancer is a common digestive system cancer, and is the third most common cause of cancer death after gastric cancer and esophageal cancer. Hepatic cancer is the second cause of cancer death in China, with approximately 110 thousand deaths per year, accounting for 45%hepatic cancer deaths in the world. The survival rate of patients with terminal hepatic cancer is very low due to the spread and metastasis of cancer cells, and recurrence rate is up to 75%after live tumor resection. The invasion and metastasis of hepatic cancer cell may be the major cause of recurrence and death and it is a complex multi-step biological process, so the metastasis mechanism of hepatic cancer is very important to the prevention and therapy.Objective:The purpose of this project is to investigate the effect of toll-like receptor (TLR) 4 activation on the invasion ability of hepatic cancer cell lines HepG2, SMMC7721and Hep3B in vitro. Furthermore, we checked the activation or phosphorylation of signaling molecules in the TLR4 signal transduction pathway, and used the inhibitor of signaling molecule to determine the role of the signal molecule in the process of tumor cells invasion. The relationship between TLR4 activation and invasion ability of hepatic cancer cells may provide potential targets for the prevention and therapy of hepatic cancer in future.Methods:TLR4 mRNA and protein levels were detected in hepatic cancer cell lines HepG2, SMMC7721 and Hep3B by real-time PCR and flow cytometry. MMP2 and MMP9 mRNA levels also were detected by real-time PCR. After treatment with 1μg/ml LPS for different time, the change of TLR4, MMP2, MMP9 mRNA expression levels were detected by real-time PCR, and TLR4 protein expression was detected by FACS. At the same time, the phosphorylation levels of JNK, IκB, NF-κB and the translocation of NF-κB from cytoplasm to nuclear were detected by Western blot after LPS stimulation. Assess the basic invasion ability of HepG2, SMMC7721, Hep3B by scratching assay and detect the changes of invasion ability of these cell lines by transwell after LPS stimulation in vitro. We slao observe whether JNK inhibitor SP600125 can inhibit the invasion ability of cells and the activation of downstream signaling molecules.Results:1. TLR4, MMP2 and MMP9 were expressed in HepG2, SMMC7721, Hep3B cells, which were detected in mRNA level or protein level.2. After LPS stimulation, the mRNA and protein levels of TLR4 increased, and the mRNA levels of MMP2 and MMP9 also increased in HepG2, SMMC7721, Hep3B cells.3. LPS stimulation enhanced the invasion ability of HepG2, SMMC7721 and Hep3B cells in vitro.4. The phosphorylation level of JNK, IκB and NF-κB increased after LPS stimulation. Moreover, the nuclear translocation of NF-κB increased. But the phosphorylation level of ERK and p38 didn't changed.5. JNK inhibitor SP600125 inhibited the invasion ability of HepG2, SMMC7721 and Hep3B cells after LPS stimulation.6. JNK inhibitor SP600125 inhibited the increase of phosphorylation level of JNK and NF-κB after LPS stimulation.Conclusion:TLR4 activation can promote the invasion ability of hepatic cancer cell.