Four human mesothelioma cell lines were used in this study. Two cell lines (ACC-MESO1 and ACC-MESO4) were purchased from the RIKEN BioResearch Center (Tsukuba, Japan) (15) and the other two (CRL-5915 and CRL-5946) were purchased from the American Type Culture Collection. Cells were cultured with Roswell Park Memorial Institute 1640 medium with GlutaMax added, containing 10% fetal bovine serum, sodium pyruvate, kanamycin, and fungizone (all purchased from Thermo Fisher Scientific). Cells were maintained in a 5% CO₂ incubator at 37°C.

The miR-18a inhibitor (mirVana; has-miR-18a-3p MH12264) and a negative control miRNA inhibitor (mirVana; negative control #1) were purchased from Thermo Fisher Scientific. Mesothelioma cells at 60–80% confluence were transfected with 50 nM of the miR-18a or negative control inhibitor using Lipofectamine RNAiMAX (Thermo Fisher Scientific) in Opti-Mem Reduced Serum Medium (Thermo Fisher Scientific) according to the manufacturer's recommended protocols.

After transfection, mesothelioma cell lines were incubated in growth medium in a 96-well plate. The proliferation rate was determined at 24, 48, and 72 h with the Cell Titer Glo 2.0 reagent (Promega KK), which measures the number of viable cells relative to their ATP level, using a GloMax Explore microplate reader (Promega) according to the manufacturer's recommended protocols.

Mesothelioma cell lines transfected with the miRNA inhibitor or control were seeded into collagen-coated 6-well plates at a density of 500 cells/well and incubated in growth medium for three weeks. Cellmatrix Type I-A (Nitta Gelatin) was used for collagen coating. Colonies were stained by Cell Stain (EMD Millipore) and counted.

The migration ability of mesothelioma cells was analyzed by a wound scratch assay. miRNA inhibitor- and control-transfected cells were incubated in collagen-coated 24-well plates. After making wounds with 1 ml micropipette tips, floating cells were removed by washing twice with fresh growth medium. Microscopic images were obtained at 0, 12, and 24 h (ACC-MESO1 cells), or at 0, 24, and 48 h (ACC-MESO4, CRL-5915, and CRL-5946 cells). The wound area was measured using TScratch software (16).

Mesothelioma cell lines were incubated with the miRNA or control inhibitor in FluoroBlok culture inserts with 8-µm pores (BD Biosciences) and coated with Geltrex Matrigel (Thermo Fisher Scientific) according to the manufacturers' protocols. Invaded cells were measured at 24 or 48 h after incubation with the miRNA inhibitor.

Mesothelioma cells were incubated with the miRNA or control inhibitor in 96-well plates for 24 h, and the RealTime Glo Annexin V Apoptosis Assay reagent (Promega) was added to the cells after transfection. Relative levels of apoptosis and necrosis were measured by analyzing luminescence and fluorescence with a GloMax microplate reader according to the manufacturer's recommended protocol.

Mesothelioma cells transfected with the miRNA or control inhibitor were seeded into 96-well plates containing 0 to 50 µM cisplatin or 0–100 µM pemetrexed (both purchased from Fujifilm Wako Pure Chemical Corporation). Viable cells were measured with the Cell Titer Glo 2.0 reagent (Promega) 72 h after the addition of chemical agents.

RNA was extracted from cells transfected with the miRNA inhibitor or control using a Maxwell RSC simplyRNA Cells kit and analyzed on a Maxwell RSC Instrument (Promega) according to the manufacturer's instructions. The extracted RNA was reverse-transcribed with SuperScript IV VILO Master Mix (Thermo Fisher Scientific) and amplified using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on an AriaMax Real-Time PCR System (Agilent Technologies). Relative expression levels were calculated according to the comparative CT (ΔΔCT) method (17). Expression levels were normalized against the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following primers were used: CDKN2D forward, 5′-TCACACTGCTGTGGTCAGCTTT-3′, reverse, 5′-AGGATGTCCACGAGGTCCTGA-3′, GAPDH forward, 5′-ACAACTTTTGGTATCATGGAAGG-3′, and reverse, 5′-GCCATCACGCCACAGTTTC-3′.

All experiments were conducted at least three times. Experimental data are presented as means ± standard deviation. Statistical significance of differences between two groups was analyzed with an unpaired Student's t-test. Statistical significance was set at P<0.05.

Inhibition of miR-18a significantly decreased proliferation of mesothelioma cells compared to that by the negative control inhibitor in all four cell lines (Fig. 1A). After 3 days, inhibition of miR-18a significantly reduced viability by 42.3% in ACC-MESO1, 33.5% in ACC-MESO4, 32.9% in CRL-5915, and 27.5% in CRL-5946 cells. The role of miR-18a in mesothelioma cell proliferation was also evaluated by the colony formation assay (Fig. 1B and C). Inhibition of miR-18a significantly reduced the colony forming ability of all four cell lines.

To understand the mechanism of miR-18a in inhibiting mesothelioma cell growth, we searched for its target gene using the online miRNA target database, Target Scan Human 7.2 (www.targetscan.org). CDKN2D was identified as a target gene of miR-18a. Furthermore, RT-qPCR analysis showed that inhibition of miR-18a significantly upregulated the expression of CDKN2D (Fig. 2).

Mesothelioma cells transfected with the miR-18a inhibitor exhibited lower migration rates compared to those transfected with the negative control inhibitor in all four cell lines (Fig. 3). At 24 h, inhibition of miR-18a reduced the migration of ACC-MESO1 cells by 41.0%, and at 48 h inhibition of miR-18a reduced the migration of ACC-MESO4, CRL-5915, and CRL-5946 cells by 50.5, 53.0, and 33.7%, respectively. Mesothelioma cell invasion was not significantly changed by inhibiting miR-18a (data not shown).

Transfection of the miR-18a inhibitor significantly increased the extent of apoptosis compared to that caused by the negative control inhibitor (Fig. 4A). Notably, ACC-MESO4 cells transfected with the miR-18a inhibitor exhibited over a three times increase in apoptosis compared to cells transfected with the negative control. However, no obvious change was observed in the extent of necrosis (Fig. 4B).

In the chemosensitivity assay, CRL-5915 cells were more sensitive to both cisplatin and pemetrexed than the other three cell lines (ACC-MESO1, ACC-MESO4, and CRL-5946). We also found that transfection with the miR-18a inhibitor significantly enhanced sensitivity to cisplatin independent of the original sensitivity (Fig. 5A). At 0.5 µM cisplatin, transfection of the miR-18a inhibitor reduced viability by 10.9, 16.0, 20.6, and 16.3% in ACC-MESO1, ACC-MESO4, CRL-5915, and CRL-5946 cells, respectively (statistically significant in ACC-MESO4, CRL-5915, and CRL-5946 cells). At 5 µM cisplatin, transfection of the miR-18a inhibitor reduced viability by 20.5, 23.3, 20.5, and 19.7% in ACC-MESO1, ACC-MESO4, CRL-5915, and CRL-5946 cells, respectively (statistically significant in all four cell lines). In this experiment, no obvious change in pemetrexed sensitivity was observed by transfection of the miR-18a inhibitor in cell lines relatively sensitive or resistant to this drug (Fig. 5B).