Role of AP-2α and MAPK7 in the regulation of autocrine TGF-β/miR-200b signals to maintain epithelial-mesenchymal transition in cholangiocarcinoma

Background: Cholangiocarcinoma (CCA) is characterized by early lymphatic, metastasis, and low survival rate. Epithelial-mesenchymal transition (EMT) is able to induce tumor metastasis. Although the TGF-beta/miR-200 signals promote EMT in various types of cancer, the regulatory mechanism in CCA is still unclear. Methods: Expression of miR-200b, TGF-beta, and EMT markers were measured in tumor samples and cell lines by qRT-PCR and western blot. CCK8 assay was performed to measure the cell viability. Transwell assay was used to evaluate migration and invasion. The target genes of miR-200b and transcription factor of TGF-beta were analyzed using dual-luciferase reporter system. Results: We have demonstrated that CCA exhibited remarkable EMT phenotype and miR-200b was reduced in CCA patients (n = 20) and negatively correlated to TGF-beta. Moreover, two CCA cells, HCCC, and RBE, with epithelial appearances treated with TGF-beta, showed fibroblastic-like cell morphology with downregulated miR-200b expression. Forced expression of miR-200b abrogated TGF-beta-induced EMT initiation, with decreased cell proliferation, migration, and invasion in vitro. Also, TFAP2A (encode AP-2 alpha) and MAPK7 were found to be targeted by miR-200b to downregulate EMT and AP-2 alpha inhibited miR-200b by directly promoting transcription of TGFB1. Overexpression of MAPK7 significantly reversed miR-200b-induced inhibition of EMT, migration, and proliferation by increasing the expression of TGF-beta, cyclin D1, and Cdk2. Further, the administration of miR-200b induced a remarkably tumor regression in vivo and reduced the effect of TGF-beta-related EMT in AP-2 alpha and MAPK7-dependent manner. Conclusions: Our study highlights that miR-200b-based gene therapy is effective in the treatment of CCA.