Fig 1: miR-3140 targeted CDK2 and EGFR by binding their 3′UTR regions. (a) Left, identification of downregulated genes after miR-3140 transfection by a gene expression array. The Venn diagram shows that 228 genes were commonly downregulated (fold change >2) by transfection of miR-3140 in Panc1, MIAPaCa2, and MDA-MB-231 cells. Right, prediction of candidate target genes regulated by miR-3140 via their 3′UTR. The Venn diagram shows that 99 genes were predicted as candidate 3′UTR-targets of miR-3140 by the TargetScan program. (b) Western blot analysis of CDK2, CDK6, and EGFR in Panc1 and MIAPaCa2 cells 72 hours after transfection with 10 nmol/L of miR-NC or miR-3140. (c) Luciferase reporter assays. Panc1 cells were transfected with the pmirGLO Dual Luciferase vectors containing wild type (Wt) CDK2 and EGFR or mutant (Mt) 3’UTR target sites of these genes, and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding site of miR-3140 within the 3′UTR of each gene and mutant sequences. Bottom, results of the luciferase assay; *P < 0.05. (d,e) Evaluation of the effect of si-CDK2 or si-EGFR. Western blot analysis (top) and cell growth assay (bottom) in indicated cell lines after transfection with 20 nmol/L of negative control siRNA (si-NC) or siRNA targeting each gene. (f) Evaluation of the effect of miR-3140 in EGFR-TKI-resistant lung cancer cells. Western blot analysis (top) and cell growth assay (bottom) in the indicated cell line after transfection with 10 nmol/L of miR-NC or miR-3140. Cell growth ratio was assessed with the WST-8 assay based on the relative ratio compared with day 1. Bar, SD for triplicate experiments; *P < 0.05.