MicroRNA miR-34b/c Enhances Cellular Radiosensitivity of Malignant Pleural Mesothelioma Cells

Materials and Methods

Cell culture and establishment of stable miR-34b/c transfectants. Two MPM cell lines (H2052 and H28) in which the expressions of miR-34b and miR-34c were epigenetically suppressed were used in this study. The two MPM cell lines were kindly provided by Dr. Adi F. Gazdar (Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA) and were maintained in RPMI-1640 (Sigma Chemical Co., Saint Louis, MO, USA) supplemented with 10% fetal bovine serum in 5% CO₂. Stable miR-34b/c and scramble RNA transfectants were established, as previously described in our report (7). Briefly, a fragment of genomic DNA encoding miR-34b/c or scramble RNA fragment as a negative control was subcloned into a pSilencer 4.1-CMV neo plasmid vector (Ambion, Austin, TX, USA). The constructed plasmids were then transfected into MPM cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). Resistant clones were isolated by G418 selection for two weeks and were maintained in G418-containing medium.

Evaluation of miR-34b/c expression using quantitative real-time-polymerase chain reaction (PCR). microRNA was extracted from miR-34b/c-transfected and scramble RNA-transfected MPM cells using TaqMan MicroRNA Cells-to-CT™ Kit (Ambion). Real-time PCR for miR-34b and 34c was performed using TaqMan MicroRNA Assays (P/N4427975, Assay ID 000427 for has-miR-34b, Assay ID 000428 for has-miR-34c; Applied Biosystems, Foster City, CA) using the StepOnePlus™ Real-Time PCR system (Applied Biosystems). The expression of miR-374 was used as an endogenous control following the manufacturer's recommendation (www.appliedbiosystems.com). The relative expression ratio for miR-34b and miR-34c was calculated from triplicate PCR.

Clonogenic cell survival assay. miR-34b/c-transfected and scramble RNA-transfected H2052 and H28 cells were trypsinized to generate a single-cell suspension, and a specified number of cells were seeded into each well in six-well tissue culture plates. After allowing the cells time to attach (6 h), the plates were irradiated at 2, 4, 6 or 8 Gy. Fourteen days after IR, colonies were stained with crystal violet. The number of colonies containing at least 50 cells was determined, and survival curves were generated. The dose enhancement factor (DEF) was calculated at a surviving fraction of 0.1 to estimate the increase in radiosensitivity. Data presented are the mean±standard deviation (SD) from at least two independent experiments.

Immunofluorescent staining for phosphorylated histone H2AX (γH2AX). γH2AX has been established as a sensitive indicator of DNA DSB with the resolution of foci corresponding to DNA DSB repair (13). miR-34b/c-transfected and scramble RNA-transfected H2052 cells were grown in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL, USA). The cells were fixed in 4% paraformaldehyde for 10 min at 1 h, 6 h, and 24 h after IR (4 Gy) and permeabilized in phosphate-buffered saline (PBS) containing 0.2% NP40 for 15 min. The cells were then incubated overnight at 4°C with 1:200 diluted anti-γH2AX antibody (Millipore, Billerica, MA,USA). The cells were incubated with 1:50 diluted fluorescein isothiocyanate (FITC)-labeled secondary antibody (Jackson Immuno Research Labs, West Grove, PA, USA) for 1 h and were then incubated in PBS, containing 4’,6-diamidino-2-phenylindole (1 μg/ml) for 30 min. Coverslips were mounted using an anti-fade solution (DAKO Corp., Carpinteria, CA, USA). The slides were examined under a fluorescent microscope (Keyence BZ-8000; Keyence, Osaka, Japan). The number of γH2AX foci was counted in each nucleus, of at least 50 cells in each sample.

Cell-cycle analysis. The cell-cycle distribution was evaluated using flow cytometry in miR-34b/c-transfected and scramble RNA-transfected H2052 cells. Non-irradiated transfectants and 4 Gy-irradiated transfectants at 24 h after IR were harvested and resuspended in PBS containing 0.2% Triton X-100 and 1 mg/ml RNase, then stained with propidium iodide and analyzed using a FACScan instrument, as previously described (7). Doublets, cell debris, and fixation artifacts were gated out, and cell-cycle analysis was performed using the CellQuest version 3.1 software.

Western blot analysis. Cells were harvested and western blot analysis was carried out as previously described (7). We examined four molecules (CCND1, CDK4, CDK6, and BCL2) reported to be primary targets of miR-34b/c. We also examined BCL2 Associated X-protein (BAX), cPARP, pro-caspase-3, and cleaved caspase-3 to detect apoptosis. Actin was used as the loading control. The primary antibodies used for western blotting were as follows: anti-CCND1 (sc718, diluted 1000:1; Santa Cruz, Santa Cruz, CA, USA), anti-CDK4 (sc260, diluted 200:1; Santa Cruz), anti-CDK6 (#3136, diluted 2000:1; Cell Signaling), anti-BCL2 (sc7382, diluted 500:1; Santa Cruz), anti-BAX (#2774, diluted 1000:1; Cell Signaling Technology, Beverly, MA, USA), anti-cPARP (#9546, diluted 2000:1; Cell Signaling), anti-pro-caspase-3 (sc7272, diluted 2000:1; Santa Cruz), and anti-actin (MAB1501, diluted 20000:1; Millipore).

Statistical analysis. Data are represented as the mean±SD. An unpaired Student's t-test was used to compare data between the two groups. Probability values less than 0.05 were considered statistically significant. All the data were analyzed using JMP9.0 for Windows (SAS Institute, Cary, NC, USA).