Patients and specimens
Tissue samples from 25 MPM patients who underwent surgery or thoracoscopic procedure in our hospital from 2007 to 2017 were collected. The diagnosis of MPM was based on the WHO criteria [11] and confirmed in all cases according to their clinical, morphologic, and immunohistochemical data. Besides, 25 normal pleura samples were collected as control from thoracoscopy-treated patients who were diagnosed with recurrent idiopathic spontaneous pneumothorax. This study was approved by the Ethical committee of Shandong Provincial Qianfoshan Hospital.
Cell culture
NCI-H2452 cells (stemming from ATCC) which belongs to human MPM cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human pleural mesothelial cell line Met-5A cells were purchased from ATCC. NCI-H2452 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1.5 g/l NaHCO3, 2.5 g/l glucose, 0.11 g/l Sodium Pyruvate and maintained in a humidified atmosphere with 5% CO2 at 37 °C. Met-5A cells were cultivated in medium 199, supplemented with 10% FBS, 20 mM HEPES, 24 mM sodium bicarbonate, 3.3 nM epidermal growth factor, 200 nM hydrocortisone, 4 mg/l insulin, 2 mM L-glutamine, and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere at 37 °C.
Transfection
Synthetic miR-182 mimics, miR-182 inhibitors, negative control (NC) miRNAs (miR-182 mimics-NC, miR-182 inhibitor-NC), small interference RNA (siRNA) targeting Numb were purchased from GenePharma Co. Ltd. (Shanghai, China). Plasmid pcDNA3.1- Numb was synthesized by GenePharma Co., Ltd. (Shanghai, China). Cells at a density of 2×105 per well were grown to 70% confluence. 1×106 cells were put in a 60-mm dish and transfected with either the empty vector(mock)or pcDNA3.1- Numb (Numb) using Lipofectamin™ 2000 (Invitrogen) according to manufacturer’s instructions. The detailed steps were the same as previously described [12]. After incubating for 48 h, the cell transfection and interference efficiency were assessed. The transfected cells were thereafter split into three sets: one was used to analyze protein expression by Western blot (WB), the other one used for cell viability assay, and the remaining one for cell apoptosis analysis.
The corresponding miRNA sequences were as follows:
miR-NC, 5′-AUAUGACGUACGUGUAACGUACUC-3′;
miR182 mimics, 5′-UUUGGCAAUGGUAGAACUCACACU-3′;
anti-miR NC, 5′-UCCGAGUGCUAUACGCUAGUAAAU-3′;
and anti-miR182 oligonucleotides, 5′-AGUGUGAGUUCUACCAUUGCCAAA-3′.
Quantitative real-time reverse transcription polymerase chain reaction
The procedure of quantitative real-time polymerase chain reaction (qRT-PCR) for detection of miRNA or mRNA was as follows. First, total RNA was extracted using TRIzol reagent (Invitrogen). cDNA was then synthesized using the TaqMan microRNA Reverse Transcription Kit (Life Technologies) for analysis of miRNA. While for the analysis of mRNA, reverse transcription was firstly carried out using ReverTra Ace qPCR RT Kit ( Toyobo, Japan ), and qRT-PCR was subsequently performed using SYBR Green Realtime PCR Master Mix (Toyobo, Japan) and solutions containing specific primers of target genes on the ABI 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA ). The expression levels of U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as control.
Western blot
48 h after transfection, cells were harvested for protein analysis via WB. Total protein was extracted using RIPA lysis buffer. Proteins were then transferred to PVDF membrane and subjected to the primary antibodies including anti- Numb (1:1000, Abcam, UK), E-cadherin, Vimentin and ZEB1 (1:500, Santa Cruz Biotechnology, USA) antibodies. Peroxidase labeled anti-mouse or anti-rabbit secondary antibodies were thereafter applied (Zhongshan Goldenbridge Biotechnology, Beijing, China). Blots were developed using enhanced chemiluminescence detection reagent (Applygen Technologies Inc., Beijing, China).
Luciferase reporter assay
WT- Numb -3′UTR pmiR-ReportTM and MUT- Numb -3′UTR pmiR-ReportTM were purchased from ABI. HEK293T cells (density, 1×104 per well) were planted in 96-well plates, incubated overnight and then transfected with WT- Numb -3′UTR pmiR-ReportTM or MUT- Numb -3′UTR pmiR-ReportTM. 24 h after the transfection, the cells were treated with miR-182 (100 ng/ml) and incubated for 48 h additionally. The luciferase activity was measured using a dual-luciferase reporter system (Promega) according to the manufacturer’s instructions.
Cell viability assay
We used MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay to evaluate cell viability. Cells with a density of 1×104 /well were seeded in a 96-well plate and incubated for 24 h. Then, these cells were treated with different concentrations of pemetrexed for 24 h in triplicate. 20 μl of 5 mg/ml MTT was added into each well and incubated for 4 h at 37 °C in culture hood. Media were carefully removed and 150 μl dimethyl sulfoxide was added. The absorbance was measured at 490 nm from which the background was subtracted. The cell survival index was calculated as [A490 (pemetrexed +)/A490 (pemetrexed −)]×100%.
Cell apoptosis analysis
The apoptosis rate was examined by double staining assays (Annexin V/FITC binding and propidium iodide (PI) uptake) using flow cytometry. 1×105cells were seeded into 6-well plates and were tested 72 h after transfection; 1×105cells were seeded into 6-well plates and incubated for another 24h, then these cells were treated with 5μmol/L pemetrexed for 24 h. After that, cells were incubated with FITC-conjugated Annexin V for 20 min at room temperature in the dark. PI was then added and the samples were immediately analyzed by fluorescence-activated cell sorting.
Transwell assay
The transwell assay was performed to assess the invasive ability of MPM cells. 24-well Boyden chambers coated with Matrigel diluted in 1:4 by precooled serum-free medium were used. The parental and transfected cells were resuspended at a density of 1 × 106/ml after starvation for 24 h. Then, 2 × 105 cells were seeded into the upper chambers, while medium supplemented with 10% FBS was placed in the lower chamber. After incubated for 24 h at 37 °C, cells on the top side of the filter were removed. The remaining cells were fixed by 4% methanol for 15 min and stained with 0.5% crystal violet for 10 min. The number of migratory cells from at least five randomly selected microscopic fields were counted and the average value was calculated. The experiments were repeated three times.
Statistical analysis
Statistical analyses were performed using SPSS 21.0 software package (SPSS Inc., Chicago, IL, USA). Data were presented as mean ± SD of three independent experiments. Student’s test, correlation analysis and analysis of variance (ANOVA) were used. All statistical tests were two-sided with probability values <0.05 defined as statistically significant.