MiR-301a transcriptionally activated by HIF-2α promotes hypoxia-induced epithelial-mesenchymal transition by targeting TP63 in pancreatic cancer

BACKGROUND: Pancreatic cancer (PC) is one of the deadliest cancers worldwide. PC metastasis involves a complex set of events, including epithelial-mesenchymal transition (EMT), that increase tumor cell invasiveness. Recent evidence has shown that hypoxia is a major EMT regulator in pancreatic cancer cells and facilitates metastasis; however, the mechanisms remain elusive. AIM: To investigate the role of miR-301a in hypoxia-induced EMT in PC cells. METHODS: Real-time PCR and Western blot analysis were used to detect the expression of miR-301a and EMT markers in PDAC cells cultured in hypoxic and normoxic conditions. Western blot analysis was used to detect the expression of EMT markers in PDAC cells with miR-301a overexpression. Wound healing assay and Transwell assay were used to detect the migration capabilities of PDAC cells with miR-301a overexpression and knockout. Luciferase assay was used to detect the miR-301a promoter and the 3’ untranslated region activity of TP63. Orthotopic PC mouse models were used to study the role of miR-301a in metastasis of PDAC cells in vivo. In situ hybridization assay was used to detect the expression of miR-301a in PDAC patient samples (adjacent paratumor and paired tumor tissues). RESULTS: Hypoxic environment could directly promote the EMT of PC cells. The expression level of miR-301a was increased in a HIF2α dependent manner in hypoxia-cultured CFPAC-1 and BxPC-3 cells. Overexpression of miR-301a enhanced the hypoxia-induced EMT of PC cells, while knocking out miR-301a result in the suppression of hypoxia-induced EMT. TP63 was a direct target of miR-301a and involved in the metastatic process of PC cells. Furthermore, miR-301a upregulation facilitated PDAC distant metastasis and lymph node metastasis in vivo. Additionally, miR-301a overexpression was indicative of aggressive clinicopathological behaviors and poor prognosis. CONCLUSION: The newly identified HIF-2α-miR301a-TP63 signaling pathway may play a crucial role in hypoxia-induced EMT in PDAC cells.