Background
Malignant pleural mesothelioma (MPM) is considered as one of the highest aggressive tumor arising from the cells lining serosal cavities, mostly resulting from the occupational exposure to asbestos fibers [
1]. Although great efforts have been made toward improving diagnosis and treatment [
2], there are no efficacious therapies for MPM patients presently, and therefore the overall survival is extremely poor. Thus, the investigation of developing the novel therapeutics, especially molecular targeting therapy, is very important for the patients with MPM.
MTA (metastasis-associated gene) is a newly discovered family of cancer progression-related genes and their encoded products. MTA are integral parts of nucleosome remodeling and histone deacetylation (NuRD) complexes, function as transcriptional co-repressors which regulate varieties pathways, including hormonal action, epithelial-to-mesenchymal transitions, differentiations, protein stability and development [
3,
4]. MTA1, the first gene found in this family, has been repeatedly reported to be overexpressed along with its protein product MTA1 in a wide range of human cancers such as endometrial adenocarcinomas, gastrointestinal carcinoids, colorectal carcinomas, hepatocellular carcinomas and non-small cell lung cancers [
5‐
10]. However, the potential prognostic relevance of MTA1 expression in MPM has not yet been investigated.
In this study, we aim to investigate the role of MTA1 in the pathogenesis of MPM and identify MTA1 could promote the metastasis of MPM cells by repressing the expression of E-cadherin.
Methods
Patients and tissue samples
MPM and corresponding adjacent tissues employed in this study were obtained from 65 consecutive patients who had de novo disease and undergone surgical resection. They were included between December 2008 and November 2013 at the First Affiliated Hospital of Soochow University (Suzhou, China) and the First Affiliated Hospital of Nanjing Medical University (Nanjing, China). The patients were followed up for a median period of 12 months (range, 3–28 months) after operation and their complete clinical data were collected. The correct diagnosis was assessed by an experienced pathologist and the staging of MPM by a clinical oncologist according to AJCC/UICC Guidelines version 7.2010 MPM. Adjacent tissue was located within 3 cm of the edge of the tumor tissue (Additional file
1: Fig. 1S). The study was approved by the Ethical Committee of the First Affiliated Hospital of Soochow University (Suzhou, China) and the First Affiliated Hospital of Nanjing Medical University (Nanjing, China), and written informed consent was obtained from the each patient.
DNA and RNA preparation
Total RNA was extracted from fresh frozen tissue specimens using TRIzol method (Invitrogen, Shanghai, China) and RNA quality was detected by NanoDrop 2000 and A260/A280 was between 1.95 and 2.05. cDNA was synthesized using reverse transcriptase kit (TAKARA, Tokyo, Japan) according to the manufacturers’ protocol.
Real-time PCR analysis
MTA1 and E-cadherin mRNA levels were measured by real-time PCR using SYBR Premix Ex Taq (TAKARA, Tokyo, Japan). MTA1 and E-cadherin transcription values were normalized against the expression of β-actin. Amplification conditions, primers, and probes sequences for MTA1 and β-actin were from the work by Zhu X et al. [
9] and for E-cadherin were the same as those in the work by Martínez-Estrada et al. [
11]. All procedures are in agreement with MIQE guidelines.
Cell culture
MSTO-211H, NCI-H2452 and 293 T cell lines (ATCC, Manassas, VA) were employed for the present study. MSTO-211H and H2452 were origin from the patients with mesothelioma and cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (Invitrogen, Carlsbad, CA), while 293 T were origin from human embryonic kidney cells and cultured in DMEM high glucose medium supplemented with 10 % fetal bovine serum. All cells were maintained in a humidified 37 °C incubator with 5 % CO2.
Lentivirus production and transduction
To generate plasmid-expressing MTA1-shRNA, double-stranded oligonucleotides were cloned into pLL3.7 vector (gifted by D. Yun Chen, Nanjing Medical University, China) and named pLL3.7-shMTA1. The sequences of MTA1-shRNA used are ccggtGACCACCGACAGATACGTG ttcaagaga CACGTATCTGTCGGTGGTCTTTTTTg. The uppercase letters represent MTA1-specific sequence, and lowercase letters represent hairpin sequences. Recombinant lentivirus was generated from 293 T cells using calcium phosphate precipitation. MSTO-211H and H2452 were transfected with lentivirus using polybrene (8 μg/ml).
Western-blotting assay
Proteins were extracted from cultured cells, quantitated using a protein assay (bicinchoninic acid [BCA] method; Beyotime, Shanghai, China). Proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane, blocked in 4 % dry milk at room temperature for 1 hour, and immunostained with primary antibodies at 4 °C over-night using anti-MTA1 (1:2000, Abcam, Cambridge, MA), anti-E-cadherin (1:1000; Abcam, Cambridge, MA), and anti-GAPDH (1:1000, Kangchen,China). The results were visualized via a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system; Thermo, Rockford, IL) and exposed in Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA).
Cell proliferation assay
Cells were seeded into 96-well plates (6.0 × 103 cells per well). Cell viability was assessed by cell-counting kit-8 assay (CCK-8, Beyotime, Shanghai, China). The absorbance of each well was read on a spectrophotometer (Thermo) at 450 nm (A450). Five independent experiments were performed in quintuplicate.
Wound-healing assay
Cells were seeded in six-well plates and cultured to confluence. Wounds of 2-mm width were created with a plastic scriber and the floating cells were washed away thrice with phosphate buffered saline (PBS). After incubation in a serum-free medium for 48 hours, cultures were observed and photos were taken under a microscope. A minimum of five randomly chosen areas was measured.
Transwell invasion assay
The invasive ability of the cells was investigated using Transwells (8-μm pore size; Corning Costar Corp, Bedford, MA) put into the 24-well plates. First, 50 μl Matrigel (50 μg/ml; BD Biosciences, San Jose, CA) was added onto each surface of the chamber, incubated for 2 hours for solidification, then the supernatant was washed away with warm PBS. MSTO-211H and H2452 were suspended in RPMI 1640 containing 2 % fetal bovine serum. A total of 100 μl of the cell suspension (5 × 104 cells) was added to the upper chamber coated with Matrigel, and 400 μl of RPMI 1640 containing 10 % fetal bovine serum was added to the lower compartment. After incubation for 48 hours at 37 °C in a 5 % CO2 humidified incubator, the Matrigel and cells on the upper surface of the filter were removed with cotton swabs and the cells that invaded into the lower surface were fixed with 2 % paraformaldehyde, stained with crystal violet. Then the filters were removed from the chambers, air-dried on the precleaned slides and applied with cover-slides using resina. Images were taken under an inverted microscope (Olympus Corp, Tokyo, Japan) at × 100 magnifications over three random fields in each well. ImageJ 1.45 s software (National Insititutes of Health, Bethesda, MD) was used for integrated optical density analysis. Each experiment was performed in triplicate.
RT-PCR array
Total RNA was extracted from MSTO-211H, MSTO-211H-shMTA1, H2452 and H2452-shMTA and reverse transcribed to cDNA. Subsequently, cDNA was amplified by PCR using 23 Super Array PCR master mix (SuperArray Bioscience, Frederick, MD) and then RT-PCR was carried out using the Human Tumor Metastasis RT2 Profiler PCR array (SuperArray Bioscience, Frederick, MD) in an ABI PRISM7900 system (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions.
Luciferase report assay
The promoter of E-cadherin was synthesized artificially and cloned into the luciferase reporter vector (Promega, Madison, WI). MSTO-211H and H2452 cells were seeded into 24-well plates and cotransfected with MTA1-siRNA (pooled siGENOME SMART pool MTA1 siRNA [50 nM per well]; Dhamacon, Chicago, IL), and luciferase reporter vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the instructions. Zero, 12 and 24 hours after incubation, cells were collected and firefly and Renilla luciferase activity was measured with the dual-luciferase reporter assay system (Promega). All results were gained through three independent experiments.
TGF-β1 Inducing EMT of MPM cells
The MPM cells were cultured in vitro and induced by transforming growth fact beta1 (TGF-β1, 5 ng/ml) for 0 h, 24 h, 48 h. The morphological characteristics of cells were observed by microscope (Olympus Corp, Tokyo, Japan) and the expression of MTA1 was detected by real-time PCR.
Immunohistochemical staining
Tissues were fixed in 4 % paraformaldehyde and cut from paraffin block to 5 μm thickness. After dewaxing with xylene and rehydration with a graded series of ethanol, the slides were heated in the autoclave for three minutes using citrate buffer (PH 6.0) and incubated with primary antibody MTA1 (1:1000, Abcam, Cambridge, MA), E-cadherin (1:1000; Abcam, Cambridge, MA) at 4 °C overnight. Blocking serum or antibody dilution buffer was prepared as Negative controls. The primary antibodies utilized were all the same as for Western blot analysis. Photographs were taken by microscope (Nikon, ECLIPSE 50i) and software NIS-Elements v4.0. Average values of integrated optical density (IOD) were obtained from five random fields per slide by using Image-Pro Plus software (v5.0). Every data was detected three times at least.
Statistical analysis
Statistical analysis was performed using GraphPad Prism (version 5.01; GraphPad Software, Inc, La Jolla, CA) statistical software. The Student’s t test and paired t test were used to analyze significance between independent groups and paired materials, respectively. The correlation test was used to analyze the correlation between MTA1 and E-cadherin. The χ2 test was used to test the significance of observed differences in proportions except when the cells size was less than 5 (Fisher’s exact tests). The significance was accepted as p value was less than 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CX conceived and designed the experiments. CX, FH wrote the article. YC and HH prepared the patient samples. CX and YC performed the experiments. WY, YY and ZS collected and analyzed the datas. All authors read and approved the final manuscript.