Abstract
Down-regulation of the miRNA miR-338-3p correlates with the invasive ability of hepatocellular carcinoma (HCC) cells. However, it is currently unclear whether down-regulation of miR-338-3p induces epithelial-mesenchymal transition (EMT), which may be the underlying mechanism governing HCC invasion. Here, we demonstrate that restoration of miR-338-3p expression via transfection of a miR-338-3p mimic reversed EMT and inhibited the motility and invasiveness of HCC cells. Conversely, silencing of endogenous miR-338-3p expression with a miR-338-3p-specific inhibitor induced EMT and enhanced HCC cell motility. Additionally, Snail1 (an upstream regulatory protein of EMT) and Gli1 (a key transcription factor in the sonic hedgehog (SHH) signaling pathway) expression was up-regulated in cells treated with the miR-338-3p inhibitor and down-regulated by the miR-338-3p mimic. Further analyses demonstrated that miR-338-3p inhibitor-induced EMT in HCC cells was blocked by treatment with a small interfering RNA (siRNA) targeting Snail1, that the SHH signaling pathway was required for both miR-338-3p inhibitor-induced EMT and up-regulation of Snail1, and that miR-338-3p targeted a sequence within the 3′-untranslated region of N-cadherin mRNA. Notably, miR-338-3p expression was significantly down-regulated in HCC samples from patients with metastases and was associated with poor metastasis-free survival rates. Lastly, correlations between the expression levels of miR-338-3p and E-cadherin, Smoothened (SMO), Gli1, Snail1, N-cadherin, and vimentin were confirmed in HCC xenograft tumors and HCC patient specimens. Our findings suggest that miR-338-3p suppresses EMT and metastasis via both inhibition of the SHH/Gli1 pathway and direct binding of N-cadherin. miR-338-3p is a potential therapeutic target for metastatic HCC.
Highlights
Hepatocellular carcinoma (HCC) is one of the most common malignancies in China, and increasing incidences have been reported worldwide [1]
A dramatic morphological change was observed in MHCC-97H cells after treatment with miR-338-3p mimics; the spindlelike fibroblastic morphology was replaced by a typical cobblestone-like appearance (Figure 2A), suggesting that artificial overexpression of miR-338-3p induced mesenchymal-to-epithelial transition (MET), a reversal of the epithelial-mesenchymal transition (EMT) process
These results further suggest that down-regulation of miR-338-3p in HCC is associated with the up-regulation of SMO, the activation of the sonic hedgehog (SHH) signaling pathway and Snail1, and the promotion of metastasis in HCC through induction of EMT
Summary
Hepatocellular carcinoma (HCC) is one of the most common malignancies in China, and increasing incidences have been reported worldwide [1]. Our previous study demonstrated that miR338-3p suppresses the invasiveness and metastasis of HCC cells by directly targeting the Smoothened (SMO) protein [13], a seven-pass transmembrane protein that is widely considered a marker of activation for the sonic hedgehog (SHH) signaling pathway [14]. Inaguma et al (2011) demonstrated that activation of the SHH signaling pathway induces EMT through the inhibition of E-cadherin expression, leading to invasion and metastasis in pancreatic cancer cells [15]. Zhan et al (2014) reported a positive correlation between Gli and Snail expression levels in progressive gastric cancer [16]. The correlation between the SHH signaling pathway and miR-338-3p expression in HCC is not fully understood
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