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

World J Gastroenterol. 2020 May 21;26(19):2349-2373. doi: 10.3748/wjg.v26.i19.2349.

Abstract

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.

Keywords: Epithelial-mesenchymal transition; HIF-2α; Hypoxia; MiR-301a; Pancreatic cancer; TP63.

MeSH terms

  • 3' Untranslated Regions / genetics
  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Biomarkers, Tumor / analysis
  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / metabolism
  • Carcinoma, Pancreatic Ductal / diagnosis
  • Carcinoma, Pancreatic Ductal / genetics*
  • Carcinoma, Pancreatic Ductal / mortality
  • Carcinoma, Pancreatic Ductal / pathology
  • Cell Hypoxia / genetics
  • Cell Line, Tumor
  • Epithelial-Mesenchymal Transition / genetics
  • Female
  • Gene Expression Regulation, Neoplastic
  • Gene Knockout Techniques
  • Humans
  • Kaplan-Meier Estimate
  • Male
  • Mice
  • MicroRNAs / analysis
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Middle Aged
  • Pancreas / pathology
  • Pancreatic Neoplasms / diagnosis
  • Pancreatic Neoplasms / genetics*
  • Pancreatic Neoplasms / mortality
  • Pancreatic Neoplasms / pathology
  • Prognosis
  • Promoter Regions, Genetic / genetics
  • Signal Transduction / genetics
  • Transcription Factors / genetics*
  • Tumor Suppressor Proteins / genetics*
  • Xenograft Model Antitumor Assays

Substances

  • 3' Untranslated Regions
  • Basic Helix-Loop-Helix Transcription Factors
  • Biomarkers, Tumor
  • MIRN301A microRNA, human
  • MicroRNAs
  • TP63 protein, human
  • Transcription Factors
  • Tumor Suppressor Proteins
  • endothelial PAS domain-containing protein 1