Functional activity of miR-124a and miR-181a on target genes.

<p>(A) Schematic image of miR-124a putative target site in mouse Dlx5 3′-UTR and alignment of mir-124a with wild-type (WT) and mutant (MUT) 3′-UTR regions of Dlx5 showing complementary pairing. Mutated nucleotides are underlined. These seed regions are evolutionally well-conserved among higher vertebrates. (B) Schematic image of miR-181 putative target site in the mouse Msx2 mRNA 3′-UTR and alignment of miR-181a with wild-type (WT) and mutant (MUT) 3′-UTR regions of Msx2. Complementary pairing between miR-181a and Msx2 is shown. Mutated 3′-UTR nucleotides are underlined. These seed regions are evolutionally well-conserved among higher vertebrates. (C) MC3T3 cells were cotransfected with luciferase reporters carrying wild-type Dlx5 3′-UTR or mutated Dlx5 3′-UTR, phRL-null (Renilla plasmid), and 50 nM RNA oligonucleotides of mir-Control (mir-C) or miR-124a. The effects of miR-124a and control miRNAs on reporter constructs after 36 h are shown. The ratio of reporter (Firefly) to control phRL-null plasmid (Renilla) in relative luminescence units is plotted. Values represent means ± SD of 3 separate wells. (D) Functional activity of the luciferase reporter plasmid carrying wild-type or mutated Msx2 3′-UTR was assessed as described above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043800#pone-0043800-g004" target="_blank">Fig. 4B</a>). Values represent means ± SD of 3 separate wells. (E) miR-124a directly targets and regulates Dlx5 and inhibits osteoblastogenesis. MC3T3 osteoblast cells were transfected with miR-124a, miRNA-Control, or transfection reagent only (Mock) at the indicated concentrations. Western blots for Dlx5 and actin (as control) were performed on total cell lysates collected at 48 h. The bands of Dlx5, Msx2 and actin were detected by ImageQuant LAS4000 (GE Healthcare UK Ltd, Little Chalfont, UK), and the intensity of bands was measured by ImageJ(<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>). Values represent means ± SD of 3 separate wells. *P<0.05, compared to miR-C. (F) MC3T3 cells were transfected with miR-181a and miR-Control as described in (E). Values represent means ± SD of 3 separate wells. *P<0.05, compared to miR-C. (G) Quantitative mRNA levels (normalized by β-actin) by Q-PCR for Dlx5, Runx2, ALP, OC and OX after 50 nM oligo transfection. Values represent means ± SD of 3 separate wells. * and **P<0.05 and P<0.01, compared to miR-C, respectively. (H) The mRNA levels of osteoblast marker genes after transfection of miR-Control and miR-181a were determined as described in (G). Values represent means ± SD of 3 separate wells.* and **P<0.05 and P<0.01, compared to miR-C, respectively.</p

Sequences of primers.

<p>Sequences of primers.</p

Altered expression of miRNAs during osteogenic differentiation of iPS cells.

<p>Total RNA of iPS cells during differentiation was extracted for miRNA microarray analysis at various time points (0, 3, 7, and 15 days). Relative fold changes in miRNAs were hierarchically clustered using Cluster and TreeView.</p

Expression of miRNAs targeting Dlx5 or Msx2 and levels of Dlx5 and Msx2 mRNA in quantitative RT-PCR.

<p>(A) Time course of miR-10a, miR-10b, miR-19b, miR-9-3p, miR-124a, and miR-181a expression in differentiated iPS cells. Quantitative RT-PCR for these miRNAs was performed. Bars represent means ± SD of 3 separate wells. (B) Time course of Dlx5 and Msx2 expression in differentiated iPS cells. Quantitative RT-PCR for osteoblast markers was performed. Values represent means ± SD of 3 separate wells.</p

Sequneces of miRNAs for real-time RT-PCR.

<p>Sequneces of miRNAs for real-time RT-PCR.</p

Effects of transfection with 6 anti-miRNAs on osteoblast differentiation of iPS cells.

<p>(A) Schematic representation of the osteoblast differentiation protocol for iPS cells which were transfected with 6 anti-miRNAs including anti-miR-124a, anti-miR-181a, anti-miR-10a, anti-miR-10b, anti-miR-19b, and anti-miR-9-3p. Transfection with 6 anti-miRNAs into mouse iPS cells was carried out on day 1 and day 8. Total RNA was harvested on day 15 for quantification of osteoblastic markers. (B) Expression of Runx2, Msx2 and osteopontin in mouse iPS cells on day 0 and on day 15 following transfection of 6 anti-miRNAs. The time course of the experiment follows the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043800#pone-0043800-g005" target="_blank">Fig. 5.A</a>. Osteoblastic differentiation was examined by osteoblastic markers such as Runx2, Msx2, Dlx5, OPN, OX and OC by real-time RT-PCR. Of these markers, Runx2, Msx2 and osteopontin were significantly increased, and the other markers were not changed. Values represent means ± SD for 3 separate wells, and the data were expressed as the fold increase of mRNA on day 15 in comparison with day 0. * and **P<0.05 and P<0.01, compared to day 0, respectively. (C) Histochemical staining for ALP and alizarin red in differentiated iPS cells which were transfected with 6 anti-miRNAs. On day 15, the iPS cells which were transfected with 6 anti-miRNAs or mock control were stained (ALP and alizarin red).</p

Osteoblast differentiation induced by BMP-4 in iPS cells.

<p>(A) Schematic representation of the osteoblast differentiation protocol for iPS cells by osteogenic cocktail. (B) Histochemical staining for ALP activity and with alizarin red in differentiated iPS cells on day 3 and day 15. EBs were grown for 15 days in the presence of Dex/h-GP/AA with BMP-4 and then stained (ALP and alizarin red). (C) Time course of osteoblast marker expression in osteoblastic differentiated iPS cells. Quantitative RT-PCR for osteoblast markers was carried out. Bars represent means ± SD of 3 separate wells.</p

Putative targets of miRNAs.

<p>Putative targets of miRNAs.</p

Identification of the Genes Chemosensitizing Hepatocellular Carcinoma Cells to Interferon-α/5-Fluorouracil and Their Clinical Significance

<div><p>The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (<em>PRKAG2</em>); transforming growth factor-beta receptor II (<em>TGFBR2</em>); and exostosin 1 (<em>EXT1</em>) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of <em>TGFBR2</em> and <em>EXT1</em> enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of <em>TGFBR2</em> and <em>EXT1</em> include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the <em>PRKAG2</em> mRNA level in HCC tissues was positively correlated with survival period, suggesting that <em>PRKAG2</em> enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that <em>PRKAG2</em> mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.</p> </div

Enhancement of IFN-α/5-FU-induced apoptosis by <i>TGFBR2</i> and <i>EXT1</i> overexpression.

<p>(A) Evaluation of nuclear condensation. The arrows indicate cells with apoptosis-specific nuclear condensation and fragmentation. (B) Measurement of intracellular caspase 3/7 activity. After adenovirus infection, cells were treated with the indicated concentrations of IFN-α and 5-FU. Activity was expressed as the fold increase relative to that at 0 h. Data are expressed as mean ± standard deviation (<i>n = </i>3). Statistical significance was determined using Student’s <i>t</i>-test. *<i>P</i><0.05 compared to LacZ at 48 h.</p