Effect Of HMGB1on Invasion And Metastasis In Hepatocellular Carcinoma Cell

Objectives: Compared the different expression of HMGB1with the key factors MMP-2,MMP-9,ICAM-1and VEGF which involved intumor invasion and metastasis in hepatocellular carcinoma cells andnormal liver cells,to investigate the role of HMGB1in primary liver cancer invasion and metastasis,providing a theoretical basis for furtherstudy for the pathogenesis of liver cancer.Method:Using Real-time PCR and Western-blot assay the expression of HMGB1,RAGE,MMP-2,MMP-9,ICAM-1and VEGF from genelevel to protein level in hepatocellular carcinoma cells HepG2and normal liver cells L02,respectively.ELISA was used to measure the MMP-2, MMP-9, ICAM-1and VEGF expression in cell supernatant ofHepG2cells and L02cells.Using flow cytometry to measure the apoptosis of HepG2and L02cell which stimulated with hydrogen peroxide (200μmol/L)in0h,24h.Results:The expression of HMGB1,RAGE,MMP-2,MMP-9,ICAM-1and VEGF in hepatocellular carcinoma cells HepG2cells comparedwith normal liver cells L02have significantly increased;In medium supernatant,protein of MMP-2,MMP-9,ICAM-1and VEGF in HepG2is higher than that in L02too.Stimulated with hydrogen peroxide (200μmol/L) after24h later,the apoptosis of HepG2is less than L02,therewas significant difference between the two group (p <0.01).Conclusion:The expression of HMGB1and the factors involvedin tumor invasion and metastasis,which including MMP-2,MMP-9,ICAM-1and VEGF in HepG2cells is higher than that in normal livercells L02, indicating that HMGB1may contribute to the development of hepatocellular carcinoma.And HMGB1also could be useful to anti-apoptosis in hepatocellular carcinoma. Objective:In the first part of the study had showed that HMGB1may be closely related to the invasion and metastasis of hepatocellular carcinoma,but the exact molecular mechanism remain unknown.Inthis part of study we forced on the changes of the factors which hadrelationship with tumor cell proliferation and migration after HMGB1stimulated.Detecting connection between HMGB1and the cytokinewhich promote tumor cells invasion to explore possible mechanismsof tumor invasion and metastasis.Providing new idea for theoretical study to hepatocellular carcinoma.Method:rhHMGB1(100μg/L) and hydrogen peroxide (200μmol/L) stimulated HepG2, cell culture for0h and24h, then be measured by flow cytometry to detect apoptosis, cell cycle.With containingHMGB1or not HMGB1(100μg/L) in serum-free culture mediumHepG2cells for24hours and the cells were used for the TranswellTMassay. The colony formation were assayed by Soft agar after the cells stimulated with rhHMGB1.The expression of MMP-2,MMP-9,ICAM-1and VEGF were measured by Real-time PCR and Western-blotafter stimulated with rhHMGB124h. In the supernatant after stimulated with rhHMGB1for24h,the expression of MMP-2,MMP-9, ICAM- 1and VEGF were measured by ELISA.MTS assay was used to measure the proliferation of HepG2which contain with rhHMGB1(100μg/L) compared with the control group after0,6,12,24,36,48and72h.Results:The results showed that after stimulated with hydrogen peroxide all the groups had apoptosis, but the apoptosis in HMGB1group significantly lower than the control group.TranswellTMassay resultshowed that the invasion and migration of HepG2had significantlyincreased after stimulated with HMGB1(p <0.01); Soft agar experimental results show that HepG2cell clonogenic was significantly increased compared with the control group after stimulated with HMGB1(p<0.01).Real-time PCR and Western-blot results all suggest that the protein levels of MMP-2, MMP-9,VEGF and ICAM-1after stimulatedwith HMGB1is enhanced, there was significant difference between two groups(P<0.01).In the culture supernatant, the expression of MMP-2,MMP-9,ICAM-1and VEGF with HMGB1stimulated has increased.From36h the proliferation of HepG2in HMGB1group had obviously increased than control group.Conclusion:HMGB1stimulated HepG2could increase the expression of factors involved in tumor invasion pathways in vitro.The upregulated expression of factors is positively correlated with the invasiveness of HepG2but negatively correlated with apoptosis.HMGB1maybe closely related to the proliferation and migration of tumor cells in HCC.