EPHA5 Enhances the Stemness of Non-small cell lung cancer Cells Through Activating the Wnt Signaling Pathway.

Abstract

Purpose: To explore the effect of erythropoietin-producing hepatocellular receptor A5 (EPHA5) on the stemness of non-small cell lung cancer cells and its molecular mechanism.

Methods: Highly-expressed EPHA5 in NCI-H460 and NCI-H1229 cells was silenced. After EPHA5 silencing, the positive expression level of cluster of differentiation 133 (CD133) in NCI-H460 and NCI-H1229 cells was detected by flow cytometry, and the expression levels of stemness markers sex determining region Y-box 2 (Sox2), Nanog, Kruppel-like factor 4 (KLF4) and octamer-binding transcription factor 4 (Oct4) in cells were detected by Western blotting.

Results: The expression level of EPHA5 in non-small cell lung cancer H460 and H1229 cells was higher than that in A549 and SPC-A1 cells. After EPHA5 silencing, the levels of CD133 and stemness markers Sox2, Nanog, KLF4 and Oct4 in H460 and H1229 cells all declined. CCK-8 assay showed that Wnt agonists at a concentration of 2.5 and 5 μm had little effect on the proliferative activity of H460 and H1229 cells. Western blotting revealed that Wnt agonists at a concentration of 5 μm could better enhance the expression of β-catenin. After treatment with Wnt agonists, the expression of CD133 in H460 and H1229 cells with EPHA5 silencing by siRNA3 was higher than that before treatment, and the expression levels of Sox2, Nanog, KLF4 and Oct4 in the above two cells were also increased compared with those before treatment. However, the levels of the above indexes were all lower after treatment with Wnt agonists than those before silencing.

Conclusion: Activating the Wnt signaling pathway can induce the increase in EPHA5 expression and enhance the stemness of non-small cell lung cancer cells.