Experimental validation of Wnt1 as a direct target gene of miRNA-148a.

(A) miRNA-148a potential binding sites and mutated sites on the 3’UTR of Wnt1. (B) 293T cells were co-transfected with plasmid vector containing either miRNA-148a or negative control (miRNA-NC) and luciferase reporter vector containing wild-type(3’UTR-Wnt1), mutant form(3’UTR-Mut) or negative control(3’UTR-NC) form of 3’UTR of Wnt1 and then assessed for luciferase reporter activity at 24 hours post-transfection. MiRNA-148a significantly reduced luciferase activities by directly bounding to Wnt1 3′-UTR. (C) Correlation analysis showed that the miRNA-148a expression was negatively related to the expression of Wnt-1 in NSCLC tissues (Coefficient = -0.276, P = 0.0004). (D) Association of miRNA-148a and Wnt1 staining scores in the same adjacent non-tumor lung tissues. (E) Protein expression of Wnt1 was reduced in A549 and H1299 cells with stable expression of miRNA-148a. β-actin was used as a loading control.