Effect Of Protease-activated Receptor 2 On Invasion And Metastasis Of Hepatocellular Carcinoma Cells

Objective:The hepatocellular carcinoma (HCC) is one of most common malignant tumors in our country, which leads to high mortality rate and recurrence rate after surgery or involvement treatment; however the pharmacological treatment is not effective. High risk of metastasis is the essential reason that causes HCC's bad prognosis. Therefore, selecting the invasion and metastasis pathway of HCC and identifying key genes in this process will be beneficial to provide potent prognostic biomarker and molecular targeted drugs against HCC. The metastasis of HCC include: multi-step sequential processes, involving to cell's adhesion, the locomotory movement, matrix-degrading ability, the cell host, tissue micro-environment and many other kinds of regulation factors. Proteinase-actived receptors 2(PAR-2) is a G protein-coupled seven-trans-membrane-domain receptor, the trypsin and tryptase are natural agonists of this receptor, artificial synthesis of small molecule polypeptide just like SLIGKV-NH2 can also activate this receptor. Some researches showed the expression level of PAR-2 is closely related to the multiplication and metastasis of many kinds of tumor cells. Our previous research demonstrated that PAR-2 express in hepatocarcinoma cell line HepG2, SMMC-7721. PAR-2 can be activated by trypsin and SLIGKV-NH2 in HepG2, thereby promoting the proliferation and induce DNA synthesis and cell division of HepG2 cells. In this experiment, we aimed to investigate the effect of PAR-2 on invasion, metastasis and the possible molecular mechanisms in HepG2 and SMMC-7721, which was activated by endogenous agonists trypsin and synthetic agonists SLIGKV-NH2.Methods: PAR-2 natural agonist trypsin and synthetic agonist SLIGKV-NH2(PAR-2-AP) were treated with HepG2 and SMMC-7721 cells. The adhesive potential was determined by cell adhesion assay on matrigel. The invasion and migration capability were examined by Transwell chamber with or without matrigel. The expression difference of VEGF-A, VEGF-C, VEGF-D, IL-8, FAK, MMP-2 and MMP-9mRNA were detected by Reverse transcription polymerase chain reaction (RT-PCR). The protein and phosphorylation activity of FAK were detected by Western blot. The gelatinase activation of MMP-2, MMP-9 was investigated by Gelatin Zymography. The VEGF and IL-8 activation were analyzed by enzyme-linked immunosorbent assay experiment (ELISA).Results:1 The adhesion experiment examined the adhesion on matrigel before and after the activation of PAR-2: Assays of adhesion on matrigel demonstrated that trypsin and SLIGKV-NH2 group enhanced cell adhesion ability after 4 hours compared with the control and anti-peptide VKGILS-NH2 groups in HepG2 and SMMC-7721 cells.2 Transwell chamber analyzed the invasion and migration before and after the activation of PAR-2: In the invasion experiments, trypsin and SLIGKV-NH2 increased the number of HepG2 and SMMC-7721 cells that damage matrigel through the Polycarbonate membrane than that of anti-peptide VKGILS-NH2 and control groups. Statistically, the difference is significant. In the migration experiments, PAR-2, which were activated by trypsin and SLIGKV-NH2, significantly increase the number of HepG2 and SMMC-7721 cells resulted from deformation of movement through the Polycarbonate membrane than the corresponding numbers performed by anti-peptide VKGILS-NH2 and control groups.3 The expressions of VEGF and IL-8 before and after the activation of PAR-2 were investigated by RT-PCR and ELISA: First, trypsin and SLIGKV-NH2 groups increase the expression VEGF-A and IL-8 without VEGF-C and VEGF-D mRNA by RT-PCR in comparison with the control and anti-peptide VKGILS-NH2 groups in HepG2 and SMMC-7721 cells. We found that the secretion capacity of VEGF-A and IL-8 tested by ELISA also showed significantly enhancement in trypsin and SLIGKV-NH2 groups. 4 The expressions of FAK before and after the activation of PAR-2 were analyzed by RT-PCR and Western blot: The RT-PCT results showed that trypsin and SLIGKV-NH2 groups increase and the expression of FAK mRNA than the anti-peptide VKGILS-NH2 and control groups in HepG2 cells. Moreover, Western blot results showed the changes of FAK phosphorylation activity showed similar trends with the RT-PCR results in HepG2 cells.5 The expressions of MMP-2 and MMP-9 before and after the activation of PAR-2 were analyzed by RT-PCR and Gelatin Zymography: The RT-PCT results showed that trypsin and SLIGKV-NH2 groups increase and the expression of MMP-2 and MMP-9 mRNA than the anti-peptide VKGILS-NH2 and control groups in HepG2 cells. Moreover, Gelatin Zymography results showed the changes of MMP-2 and MMP-9 gelatinolytic activities showed similar trends with the RT-PCR results in HepG2 without SMMC-7721cells.Conclusions:1 Trypsin and SLIGKV-NH2 can increase the adhesion, invasion and migration ability through activating PAR-2 in HepG2, SMMC-7721 cells.2 Activated PAR-2 promotes the VEGF-A's secretions of HepG2 and SMMC7721 cells, which can promote the HCC's angiogenesis.3 Activated PAR-2 promotes the IL-8's secretions of HepG2 and SMMC7721 cells, which can promote the HCC's invasion and angiogenesis.4 PAR-2 can activate the FAK, MMP-2 and MMP-9 of HepG2 cell, which can promotes the adhesion and invasion ability of HCC.