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. 2013 Oct 25;8(10):e77468. doi: 10.1371/journal.pone.0077468

Figure 5. Validation of putative miR-1207-5p target genes.

Figure 5

miR-1207-5p target genes were identified by TargetScan software and four genes were selected using Pathway Studio software based on potential biological effects relevant to ECM accumulation. The four genes selected, G6PD, PMEPA1, PDPK1, and SMAD7, were validated as putative miR-1207-5p target genes in MC using 25 nM miR-1207-5p or NC mimic (A), and 50 nM anti-miR-1207-5p or negative control (B). Results in A and B represent averages from three independent experiments. Data are means ± SD. About 20,000 human embryonic kidney 293 (HEK293) cells were seeded per well into a white 96-well plate and co-transfected with 100 ng of the indicated 3′UTR luciferase reporter vectors and 30 nM miR-1207-5p or negative control mimic (Dharmacon) using 0.2 µl per well of Lipofectamine 2000 (C). HEK293 cells were also co-transfected with reporter vectors and 50 nM miR-1207-5p inhibitor or negative control (Dharmacon) (D). Luciferase activity was measured 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to the corresponding renilla luciferase activity and plotted as a percentage of the control (HEK293 cells co-transfected with plasmid and control mimic or inhibitor). Approximately 70,000 MC/well were seeded in a 12-well plate and transfected with 3 µl Lipofectamine RNAiMAX (Life Technologies) mixed with 30 nM miR-1207-5p or negative control mimic, and 50 nM anti-miR-1207-5p or negative control. About 24 h after transfection, cells were stimulated with high glucose [30 mM] and incubated for another 24 h. Afterwards, cell culture media was removed, and cells were lysed and phosphorylathed Smad3 was quantified using phospho-SMAD3 (ser423/425) Instant One ELISA (eBiosciences), as per manufacturer's instructions (E). Experiments showed in C, D, and E were performed in quadruplicate. The significance is indicated only for samples that are significant different from all the others. * P<0.05; ** P<0.01; *** P<0.001. A.U.: arbitrary units. G6PD, glucose-6-phosphate dehydrogenase; PMEPA1, prostate transmembrane protein, androgen induced 1; PDPK1, 3-phosphoinositide dependent protein kinase-1; SMAD7, SMAD family member 7; TGF-β, transforming growth factor beta; PAI-1, plasminogen-activator inhibitor 1; MC, mesangial cells; NC, negative control; p-Smad3, phosphorylated Smad3.