Downregulation of GSK3β by miR-544a to maintain self-renewal ability of lung caner stem cells

In order to study the influence and mechanism of miR-544a on the self-renewal ability of lung cancer stem cells, TargetScan was used to predict the target gene of miR-544a. A luciferase reporter system and western blotting were used to validate the target genes identified by TargetScan. 95C and 95D low and high metastatic human lung cancer cells were transfected with miR-544a, and quantitative polymerase chain reaction (qPCR) was used to verify the miR-544a expression in these two cell lines. Tumor ball (spheroid) suspension culture was use to study the effects of miR-544a on lung cancer stem cells. TargetScan predicted that miR-544a interacted with GSK3β. A luciferase reporter system (F=201.37, P<0.01) and western blot analysis was used to validate that miR-544a could inhibit the expression of GSK3β, while β-catenin and CD133 were significantly increased in miR-544a-overexpressing 95C and 95D cells (F=9.43, 7.73 and 3.37, respectively; P<0.01). qPCR revealed that miR-544a was overexpressed in transfected 95C and 95D cells (20.51±0.97 and 15.16±1.38, respectively; F=418.05; P<0.01). miR-544a-overexpressing cells formed spheroids in suspension cultures of spheroid single cells. miR-544a was shown to reduce the expression of GSK3β and activate the Wnt signaling pathway to maintain the self-renewal ability of lung caner stem cells.