Fig 2: HNRNPC and NFIX as direct targets for miR-744. To validate miR-744 mediated regulation of potential target genes, SKOV3 and OVCAR3 cells were transfected with miR-744 mimic or non-targeting siRNA (NT, negative control for cell death) as described in Fig. 3. After 48 h, potential target gene expression was analysed by qRT-PCR. The relative mRNA expression of potential target genes was normalized to PPIA and NT employing the Livak method55 (a). Significantly downregulated potential targets were further analysed by the Luciferase Reporter Assay (b and c) to proof direct binding of the miRNA to its predicted target sequence. Anti-miR-744 was used to confirm the binding sides of miR-744 to the target mRNAs (d). The binding sites in the 3'UTR (Table 3), based on data of microRNA.org34 and TargetScanHuman35, were cloned into a pMirGLO vector. Relative luciferase activity was measured 72 h after co-transfection of the pMirGLO vector with miR-744 mimic, miRNA inhibitor anti-miR-744 or non-targeting siRNA (NT) in HEK293T cells. The relative luciferase expression was normalized to the expression after co-transfection with NT. For Western Blot analysis, SKOV3 and OVCAR3 cells were harvested 60 h after miR-744 transfection (e). The representative blots were stained with antibodies against NFIX and HNRNPC. GAPDH was used as loading control. Statistical differences were tested using paired t-test [n = 3 biological and technical replicates; mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001].