Effect of miR-34a on downstream pathway signals in HCC cells.

<p>Western blot and signal intensity of the bands. Antibodies include: phospho-AKT (p-AKT), p-ERK1/2, p-stat5 and β-actin. M: mock control; C1: Negative control for miRNA inhibitor; Inhi: miR-34a inhibitor; C2: Negative control for miRNA mimic; Mimi: miR-34a mimic.</p

Underexpression of miR-34a in Hepatocellular Carcinoma and Its Contribution towards Enhancement of Proliferating Inhibitory Effects of Agents Targeting c-MET

<div><p>Aberrant expression of microRNA-34a (miR-34a) has been reported to be involved in the tumorigenesis and progression of various classes of malignancies. However, its role in hepatocellular carcinoma (HCC) has not been completely clarified. In the current study, we have investigated the clinical significance and the <i>in vitro</i> contribution of miR-34a on biological functions of human HCCs. miR-34a expression in eighty-three cases of HCC formalin-fixed paraffin-embedded (FFPE) tissues decreased significantly compared to that in the adjacent liver tissues (<i>P</i><0.01), as detected by real-time quantitative RT-PCR (RT-qPCR). miR-34a expression in the groups of TNM stage I and II, without metastasis and without portal vein tumor embolus, was significantly higher than that of their corresponding groups (<i>P</i><0.05). In functional experiments, miR-34a mimic suppressed cell growth, migration and invasion, meanwhile it increased cellular apoptosis and caspase activity in HCC cells. miR-34a mimic also reduced phospho-ERK1/2 and phospho-stat5 signaling. In addition, miR-34a mimic enhanced the effect of cell proliferation inhibition and caspase activity induction of agents targeting c-MET (siRNAs and small molecular inhibitor su11274). In conclusion, miR-34a may act as a tumor suppressor miRNA of HCC. The strategies to increase miR-34a level might be a critical targeted therapy for HCC in future.</p> </div

Relationship between miR-34a expression and clinicopathological parameters. miR-34a expression was determined using real time RT-qPCR.

<p>Relationship between miR-34a expression and clinicopathological parameters. miR-34a expression was determined using real time RT-qPCR.</p

Protein level of c-MET after treatment of siRNAs or small molecular inhibitor su11274.

<p>HepG2 cells (2.5×10⁴ cells per well using 24-well-plate) were transfected with c-MET siRNAs or treated with c-MET small molecular inhibitor su11274 for 96 h. c-MET protein level was determined using western blot. c-MET and β-actin western blot signals were quantified, and the c-MET signal intensity relative to the β-actin was calculated. These values are represented by the bar graph. M1:Mock1, mock control for siRNA containing only transfection reagent; M2: Mock2, mock control for su11274 with only 0.1% DMSO. Si: c-MET siRNAs. Su: su11274.</p

Effect of miR-34a on cell growth in HCC cells.

<p>HepG2, HepB3 and SNU449 cells (2.5×10³ cells per well in 96-well-plate) were cultured for one day and then transfected with miR-34a inhibitor, miR-34a mimic and their controls (200 nM). (A) Time dependent effect detected by CellTiter-Blue Cell Viability assay. (B) Cell proliferation assessed with MTS assay. * <i>P</i><0.05, ** <i>P</i><0.01, compared to blank and negative controls at the same time point. TOX: TOX positive transfection control.</p

miR-34a enhanced the growth inhibitory effect of c-MET targeting agents in HCC HepG2 cells.

<p>HepG2 cells (2.5×10³ cells per well in 96-well-plate) were treated with miR-34a mimic with c-MET siRNA (A) or su11274 (B). MTS was performed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061054#pone-0061054-g002" target="_blank">Figure 2</a> and the proliferation inhibition rate was calculated. Bliss independence criterion was performed to calculate the theoretical additive effect. (C) Caspase-3/7 activity. * <i>P</i><0.05, ** <i>P</i><0.01, compared to agent alone. (D) c-MET protein level with single or dual treatments detected by western blot. Antibodies include: c-MET and β-actin.</p

Effect of miR-34a on apoptosis in HCC cells.

<p>HepG2, HepB3 and SNU449 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061054#pone-0061054-g002" target="_blank">Figure 2</a>. (A) Caspase-3/7 activity was detected using Apo-ONE Homogeneous Caspase-3/7 Assay. * <i>P</i><0.05, ** <i>P</i><0.01, compared to blank and negative controls at the same time point. (B) Hoechst 33342/PI double fluorescent chromatin staining with different transfections for 96 hrs in HepG2 cells (×200). (C) Western blot and signal intensity of the bands. Cells were treated for 96 h. Antibodies include: full length caspase-3, cleaved caspase-3 and β-actin. M: mock control; C1: Negative control for miRNA inhibitor; Inhi: miR-34a inhibitor; C2: Negative control for miRNA mimic; Mimi: miR-34a mimic.</p

siRNAs used in the study.

<p>NA: not applicable.</p

Effect of miR-34a inhibitor and miR-34a mimic on cell migration and invasion in HepG2 cells.

<p>HepG2 cells (5×10⁴ cells per well using 6-well-plate) were cultured to 50% confluent and transfected with the miR-34a inhibitor, miR-34a mimic and the controls. The cells were incubated in serum-free medium for another one day 96 h post transfection. Then the cells were trypsinized and added (5×10⁴ cells) to the chamber (6.5 mm in diameter, 8 µm pore size) for both migration and invasion assays. migrating and invasive cells were fixed, stained, counted and the ratio of cell migration and cell invasion was calculated.</p

Number of putative pseudogenes.

<p>Note: Only those putative pseudogenes that score over 200 are counted, with details are presented in S-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g002" target="_blank">Fig. 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g004" target="_blank">4</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g005" target="_blank">5</a>.</p