Fig. 5

Regulation of CPNE1 expression by miR-335-5p and downregulation of miR-335-5p expression in NSCLC. a Schematic diagram showing the subcloning of the predicted miR-335-5p-binding site at positions 137–143 of the CPNE1 3′-UTR into a psiCHECK-2 luciferase construct. Predicted duplex formation between miR-335-5p and the wild-type or mutant miR-335-5p-binding site is indicated. b-c Luciferase activity of the construct containing the wild-type or mutant CPNE1 3′-UTR reporter gene in A549 and H1299 cells co-transfected with the negative control (NC) or miR-335-5p. Scrambled sequences were used as the NC. Relative Renilla luciferase activity was determined and normalized against firefly luciferase activity. d-f Expression of miR-335-5p and CPNE1 in NSCLC cells transfected with miR-335-5p mimics was detected by qRT-PCR and western blot analysis respectively. g Relative miR-335-5p levels in 60 NSCLC tissues (T) and paired noncancerous lung tissues (N). h A public Gene Expression Omnibus dataset (GSE36681) containing 47 NSCLC tissues and 47 normal lung tissues showed that miR-335-5p expression was downregulated in human NSCLC tissues. i qRT-PCR analysis of relative miR-335-5p expression in human NSCLC cell lines. *P < 0.05; **P < 0.01; ***P < 0.001