Fig. 7

MiR-654-5p is a negative regulator of C12orf59. a Illustration of screening miRNAs regulating C12orf59: Method #1 used the prediction algorithm of TARGETSCAN identified miRNAs, which hold potential as C12orf59 suppressors. Method #2 used miRNA profiling and identified miRNAs, which were downregulated significantly in GC. Then, we overlapped the results of two methods and singled out miR-654-5p as a potential regulator. b Quantitative RT-PCR assay of miR-654-5p expression in six GC cell lines (left panel), and in eight pairs of matched GC (T) and adjacent non-neoplastic gastric tissue (ANT) (right panel). c Predicted miR-654-5p target sequence in 3′-UTR of C12orf59 (C12orf59 3′-UTR) and mutant miRNA containing three altered nucleotides in the seed sequence of miR-654-5p (miR-654-5p-mut). d Luciferase assay of pGL3- C12orf59 3′-UTR reporters co-transfected with increasing amounts (10, 20 and 50 nM) of miR-654-5p mimic or miR-654-5p-mut in MNK-45 and AGS cell lines. e Western blotting analysis demonstrates that miR-654-5p transfection markedly decreased C12orf59 protein levels of MNK-45 and AGS cells, whereas miR-654-5p-mut exerted no inhibition effect. *P < 0.05