Lentiviral-mediated Smad4 RNAi promotes SMMC-7721 cell migration by regulation of MMP-2, VEGF and MAPK signaling.

Abstract

Hepatocellular carcinoma (HCC) is a highly malignant cancer characterized by rapid progression, easy metastasis and frequent recurrence. Previous studies have shown that the Smad4 signaling pathway plays an important role in the cell growth and apoptosis of HCC. However, the effect of Smad4 signaling on the invasion and migration of HCC cells remains unclear. The present study aimed to examine the effects of the transforming growth factor (TGF)-β1-Smad4 signaling pathway on the migration of HCC cells. Lentiviral vectors expressing miRNA against Smad4 were constructed to block the expression of Smad4 in HCC cells, and transwell units were used to investigate the invasive potential of SMMC-7721 cells before and after TGF-β1 treatment. mRNA levels of matrix metalloproteinase (MMP)-2 and -9 were analyzed by reverse-transcription PCR, and concentrations of vascular endothelial growth factor (VEGF), p-JNK, p-p38 and p-Erk1/2 proteins were analyzed by Western blotting. The results indicate that TGF-β1 induced cellular invasion in the SMMC-7721 cells. These effects were almost completely blocked by the knockdown of Smad4. Reverse-transcription PCR and Western blot analysis revealed that MMP-2, VEGF, p-JNK and p-p38 were up-regulated by the silencing of Smad4, while the expression of MMP-9 and p-Erk1/2 was not affected by Smad4 silencing with or without TGF-β1 stimulation. These findings suggest that TGF-β1-induced SMMC-7721 cell invasion by the up-regulation of MMP-2 and VEGF is Smad4-dependent. The activation of MMP-2 and VEGF may be an important mechanism by which Smad4 is involved in metastasis. TGF-β1-Smad4 signaling may regulate SMMC-7721 cell migration through the activation of the MAPK pathway.