Fig. 3

miR-296-5p directly targets NRG1. (a) Intersection of three profiles, including upregulated mRNAs in high metastatic cells compared with low metastatic cells, predicted targets of miR-296-5p separately by miRanda and TargetScan. (b) Schematic diagram of the dual luciferase miRNA target reporter vector, the wild type and mutant NRG1 3′-UTR sequences are shown with the miR-296-5p sequence. (c) Luciferase assays in HEK-293 T cells with wild-type or mutant NRG1 3′-UTR vectors and miR-296-5p mimic or NC. (d) qRT-PCR analysis of NRG1 mRNA levels in miR-296-5p-KD Huh7, miR-296-5p-OE MHCC97H cells and their negative control cells. (e) Western blot analysis of NRG1 and secreted NRG1 protein levels in miR-296-5p-KD Huh7, miR-296-5p-OE MHCC97H cells and their negative control cells. (f) Representative images of IHC staining of NRG1 in tumor tissues of HCC patients with high or low level of miR-296-5p (left), and an inverse correlation between the levels of NRG1 and miR-296-5p in HCC tissues (right). Magnification 100×. **p < 0.01, ***p < 0.001