Knockdown of β-Catenin Through shRNA Cause a Reversal of EMT and Metastatic Phenotypes Induced by HIF-1α

Obective: Wnt/beta-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in Epithelial-Mesenchymal transition (EMT), however, there was no evidence that Wnt/beta-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1 alpha (HIF-1 alpha) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of beta-catenin protein increased in LNCaP/HIF-1 alpha cells, but not in PC-3/HIF-1 alpha. This study mainly aimed at exploring the essentiality and importance of Wnt/beta-catenin signaling pathway in HIF-1 alpha-induced EMT. Methods: Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1 alpha. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1 alpha was transiently transfected with beta-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/beta-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay. Result: We successfully established prostate cancer cell line LNCaP/HIF-1 alpha and LNCaP/HIF-1 alpha/beta-catenin(-). LNCaP/HIF-1 alpha displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1 alpha, the epithelial marker E-cadherin was increased in LNCaP/HIF-1 alpha/beta-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through beta-catenin shRNA cause a reversal of EMT induced by HIF-1 alpha in human prostate cancer. Conclusion: Overexpression of HIF-1 alpha stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/beta-catenin signaling pathway played a vital role in this process. Wnt/beta-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1 alpha.