Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma

Figure 1 Expression profile of microRNA-17-5p and insulin-like growth factor binding protein-3 and their correlation in liver tissues. The expression of miR-17-5p and IGFBP-3 were investigated in 10 healthy and 23 HCC liver tissues using TaqMan qRT-PCR and normalized in each sample to RNU6B endogenous control for miR-17-5p and B2M for IGFBP-3. A: miR-17-5p expression was down-regulated in non-metastatic HCC patients compared to healthy liver tissues (P = 0.0012); B: Regarding IGFBP-3, its mRNA expression showed a significant higher expression in HCC tissues compared to healthy tissues (P = 0.0041). Statistical analysis was performed using the Mann-Whitney test; C: Relative quantitation (RQ) values of miR-17-5p and IGFBP-3 mRNA in HCC tissues were analyzed using Pearson’s method of correlation. A non-significant inverse correlation was found with Pearson’s r = -0.3244 (P = 0.1310). ^bP < 0.01. HCC: Hepatocellular carcinoma; IGFBP-3: Insulin-like growth factor binding protein-3; miR-17-5p: MicroRNA-17-5p; qRT-PCR: Real-time quantitative PCR.

Figure 2 Impact of microRNA-17-5p on insulin-like growth factor binding protein-3 mRNA expression in HuH-7 cell line. A: The expression of miR-17-5p was determined by TaqMan qRT-PCR in HuH-7 cells transfected with oligonucleotide mimics of miR-17-5p, 48 h post-transfection, relative to their expression in mock untransfected HuH-7 cells. The expression of miR-17-5p was normalized to RNU6B endogenous control. A: Transfection of miR-17-5p mimics increased miR-17-5p levels in HuH-7 by 250 fold compared to mock cells (P = 0.0470). Unpaired t-test was performed; B: HuH-7 cells were transfected with miR-17-5p mimics or inhibitors, and the relative expression of IGFBP-3 was determined using TaqMan qRT-PCR, relative to mock untransfected cells, and gene expression was normalized to endogenous control B2M. IGFBP-3 mRNA expression was dramatically suppressed upon mimicking of miR-17-5p compared to mock cells (P = 0.0267), while inhibitors of miR-17-5p showed a tendency of increase compared to mock cells. Unpaired t-test was performed. ^aP < 0.05. IGFBP-3: Insulin-like growth factor binding protein-3; miR-17-5p: MicroRNA-17-5p; qRT-PCR: Real-time quantitative PCR.

Figure 3 Impact of microRNA-17-5p on free insulin-like growth factor-II protein in HuH-7 cells. HuH-7 cells were transfected with miR-17-5p mimics or inhibitors. The free IGF-II protein was measured in media of mimicked and antagonized HuH-7 cells using an IGF-II ELISA Kit. Free IGF-II protein, measured at λmax = 450, was found to be significantly increased upon mimicking of miR-17-5p expression compared to mock untransfected cells (P = 0.0339), while inhibitors of miR-17-5p showed no effect on the levels of free IGF-II protein levels compared to mock cells. Unpaired t-test was performed. ^aP < 0.05. IGF-II: Insulin-like growth factor-II; miR-17-5p: MicroRNA-17-5p.

Figure 4 Insulin-like growth factor binding protein-3 is a direct target of miR-17-5p. For each target sequence, experiments were performed by transfecting HuH-7 cells with either empty pmiRGLO vector, or the construct with the wild-type (WT) miR-17-5p target region insert, or the construct with the mutant (MUT) miR-17-5p target region insert. Then miR-17-5p mimics were co-transfected with the vectors or constructs. A: Luciferase activity was not affected in cells co-transfected with miR-17-5p mimics and WT1 construct compared to cells transfected with the WT1 construct alone; B: On the other hand, luciferase activity was inhibited by 27.5%, in cells co-transfected with miR-17-5p mimics and WT2 construct compared to cells transfected with the WT2 construct alone (P = 0.0474). The cells transfected with either of the mutant constructs (MUT1 or MUT2) show no change in the luciferase activity upon mimicking with miR-17-5p. Unpaired t-test was performed. ^aP < 0.05. NS: Not significant; IGFBP-3: Insulin-like growth factor binding protein-3; miR 17-5p: MicroRNA-17-5p.