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

Background: The transforming growth factor beta (TGF-beta) activates JNK, phosphorylates Smad3 to linkerphosphorylated 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. (C) 2021 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.