54910

SEMA4C

BACKGROUND: The p38 mitogen-activated protein kinase (p38 MAPK) is an important intracellular signal transduction pathway involved in TGF-beta1-induced epithelial-mesenchymal transition (EMT). Sema4C, a member of the semaphorin family, was found to be essential for the activation of p38 MAPK. However, the role of Sema4C in promoting TGF-beta1-induced EMT is unclear. METHODS: Renal fibrosis was induced by 5/6 subtotal nephrectomy rat model. In vitro, Sema4C was induced in human proximal tubular epithelial cells (HKC) by treatment with TGF-beta1, or was inhibited by siRNA or was over-expressed by Sema4C transfection. The selective p38 MAPK inhibitor, SB203580, was administered to inhibit the p38 pathway. The expression of Sema4C, the markers of EMT, p38 phosphorylation and fibronectin secretion were measured by western blotting, immunohistochemistry, immunocytochemistry or enzyme-linked immunosorbent assay. RESULTS: The expression of Sema4C increased in HKC cells that were treated with TGF-beta1. Knockdown of Sema4C potently inhibited phosphorylation of p38 MAPK and reversed TGF-beta1-induced EMT. Over-expression of Sema4C via Sema4C transfection elicited p38 MAPK phosphorylation and promoted EMT. The effects of Sema4C during EMT were blocked by a p38-specific inhibitor. In vivo, the expression of Sema4C increased in the tubular epithelia of 5/6-nephrectomized rats and human fibrotic renal tissue, and similar localization of phosphorylated p38 and Sema4C was demonstrated by immunohistochemistry on serial sections. CONCLUSIONS: Our findings suggest that Sema4C plays an important role in TGF-beta1-induced EMT through activation of p38 MAPK in proximal tubular epithelial cells.

Erbin, a member of Leucine-rich repeat and PDZ-containing protein family, was found to inhibit TGF-beta-induced epithelial-mesenchymal transition (EMT) in our previous study. However, the mechanism of Erbin in regulating EMT is unclear. Semaphorin protein Sema4C, with PDZ binding site at C-terminal has been recognized as a positive regulator of EMT. Here, we aimed to examine the interaction between Erbin and Sema4C. HK2 cells were treated with TGF-beta1, or transfected with Erbin and (or) Sema4C. Interaction of Erbin and Sema4C was identified by immunoprecipitation. RT-PCR was used to detect the expression of Erbin and Sema4C at mRNA level after transfection. The expression levels of Erbin, Sema4C, and markers of EMT were measured by using Western blotting or ELISA. After HK2 cells were stimulated with 10 ng/mL TGF-beta1 for 72 h, the protein expression levels of Erbin and Sema4C were both up-regulated, and immunoprecipitation results showed Erbin interacted with Sema4C in HK2 cells both at endogenous and exogenous levels. Furthermore, overexpression of Sema4C suppressed E-cadherin, induced vimentin and promoted fibronectin secretion, indicating Sema4C promotes the process of EMT. However, HK2 cells overexpressing Erbin were resistant to Sema4C-induced EMT. In contrast, Erbin specific siRNA promoted EMT induced by Sema4C. Taken together, these results suggest that Erbin can interact with Sema4C, and co-expression of Erbin blocks the process of Sema4C-induced EMT.

Acquisition of epithelial-mesenchymal transition (EMT) has recently been proposed as an important contributor of drug resistance in cervical cancer cells. However, the underlying mechanisms are still unclear. MicroRNAs play a crucial role in regulating EMT. The aim of this study was to explore the potential role of miR-25-3p in regulating EMT in cisplatin-resistant (CR) cervical cancer cells. To this end, we established stable CR cervical cancer cells, HeLa-CR and CaSki-CR, and investigated the function of miR-25-3p in regulating EMT. It is found that CR cervical cancer cells possessed more EMT characteristics and demonstrated higher migratory abilities and invasiveness. miR-25-3p downregulation was also seen in HeLa-CR and CaSki-CR cells. Of note, ectopic expression of miR-25-3p reversed the EMT phenotype and sensitized CR cells to cisplatin via targeting Sema4C. Furthermore, stable overexpression of miR-25-3p in HeLa-CR cells suppressed tumor growth in mice, downregulated Sema4C and Snail, and upregulated E-cadherin compared with the control group. These results suggest that miR-25-3p is an important regulator of cervical cancer EMT and chemoresistance. Thus, upregulation of miR-25-3p could be a novel approach to treat cervical cancers that are resistant to chemotherapy.CI - (c) 2016 The Authors. cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese cancer Association.