Phospho-Smad3L promotes progression of hepatocellular carcinoma through decreasing miR-140-5p level and stimulating epithelial-mesenchymal transition

Abstract

Background: The transforming growth factor β (TGF-β) activates JNK, phosphorylates Smad3 to linker-phosphorylated Smad3 (pSmad3L), resulting in liver tumorigenesis. However, the effect of pSmad3L on hepatocellular carcinoma (HCC) prognosis is obscure.

Aim: To detect the effect of pSmad3L on HCC prognosis and investigate the mechanism.

Methods: The expressions of pSmad3L, E-cadherin, vimentin and MicroRNA-140-5p (miR-140-5p) were detected by using immunohistochemistry, immunofluorescence and in situ hybridization. Next, the relationships of pSmad3L and HCC patients' prognoses, pSmad3L and EMT markers, pSmad3L and miR-140-5p were analyzed using Spearman's rank correlation test. JNK/pSmad3L specific inhibitor SP600125 or Smad3 mutant plasmid was used to suppress JNK/pSmad3L pathway, and QPCR assay was performed to investigate the effect of pSmad3L on miR-140-5p level. The proliferation and invasion of hepatoma cells were observed using colony formation assay and transwell assay.

Results: We demonstrated that patient with high level of pSmad3L predicted poor prognosis. Next, we verified that pSmad3L promoted EMT of hepatoma cells in vivo and in vitro. In order to investigate the mechanism, we verified a negative correlation between pSmad3L and miR-140-5p, which was an EMT inhibitor, in the liver tissues of HCC patient and diethylnitrosamine (DEN)-induced rat HCC model. We further used SP600125 or pSmad3L mutant plasmid to decrease pSmad3L level of hepatoma cells, and inhibition of pSmad3L increased miR-140-5p level and suppressed EMT of hepatoma cells.

Conclusions: JNK/pSmad3L pathway induces EMT by inhibiting miR-140-5p in HCC progression.

Keywords: Epithelial-mesenchymal transition; Hepatocellular carcinoma; Linker-phosphorylated Smad3; MircoRNA-140-5p.