Background: We have reported previously that overexpression of glucose-regulated protein 78 (GRP78) promotes the invasion of hepatocellular carcinoma. However, whether GRP78 knockdown affects the extracellular matrix degradation has not been elucidated. Here we are going to determine whether GRP78 knockdown affect the ECM degradation and the role of MMP-2 and MMP-9 in these process in hepatocellular carcinoma cells.
Methods: Human hepatocellular carcinoma cell line SMMC7721 and HepG2 were cultured in DMEM supplemented with 10% FBS, RT-PCR and western blot were used to detect the endogenous expression of GRP78, MMP-2, MMP-9 and TIMP-2 in SMMC7721 and HepG2. GRP78 shRNAs were transfected using lipofection2000. Transwell assay and wound healing assay were used to analyze the invasion of each transfectant. Gelatin zymography and FITC-gelatin degradation assay were employed to investigate the capabilities of ECM degradation of each transfectant. MTT assay was used to determine the proliferation status. Western blot was employed to detect the expression of matrix metalloproteinase 2(MMP-2), MMP-9, MMP-14, and tissue inhibitor of metalloproteinases 2(TIMP-2), focal adhesion kinase (FAK), ERK1/2, JNK and Src.
Results: According to the expression levels of GRP78, MMP-2, MMP-9, MMP-14 and TIMP-1 in hepatocellular carcinoma cell lines SMMC7721 and hepG2, we used SMMC7721 as the in vitro invasion model for further functional analysis. Using this model, we found that GRP78 knockdown decreased the invasion of tumor cells, and this inhibitory effect was independent of cell proliferation. In hepatocellular carcinoma cells, Grp78 knockdown inhibited ECM degradation and the decreased activity and expression of MMP-2, but not MMP-9 contributed largely to this impact. Further analysis revealed that the decreased activity and expression of MMP-2 is mediated by JNK.
Conclusion: Knockdown of GRP78 decreases ECM degradation, and downregulates the expression and activity of MMP-2 and TIMP-2. These results further demonstrate that GRP78 is a potential target for inhibiting the invasion of hepatocellular carcinoma cells.