Cell lines and culture
The eight K30, K450, K180, K150, TE-1, K510, K140 and K410 cell lines come from our laboratory. All cell lines were cultured in RPMI1640 (Biological Industries, Israel) +10% fetal bovine serum (Invitrogen, USA) and 1% glutamine at 37 °C in 5% CO2.
Bisulfite sequencing PCR (BSP) analysis
Genomic DNA was isolated by a standard phenol/chloroform purification method, verified by electrophoresis on an agarose gel, and treated by an ammonium bisulfite-based bisulfite conversion method. Then the PCR fragments from the converted DNA were sequenced and analyzed. Raw sequence data files were processed, and the area ratio (%) of C over C + T of the primary CpG dinucleotide was calculated as the % of methylation and plotted [13].
Transient transfection assays and reagents
siRNA and scrambled (negative control, NC) sequences as well as a riboFECT CP transfection kit were supplied by Guangzhou RiboBio, China. A GFP-tagged PON3 overexpression construct (pReciever-M98) was purchased from Genecopia, Guangzhou, China (Catalog No.: EX-E0804-M98-5). Transfections of the above mentioned ribonucleic acid reagents and reporter plasmids were performed according to the manufacturer’s instructions.
Chemoresistance profiling (IC50 determination)
All of the chemotherapeutic drugs used in this study were of clinical grade. To perform thiazolyl blue tetrazolium blue (MTT)-based cell proliferation assays, experimental groups of cells in the logarithmic phase of growth were seeded in triplicate in 96-well plates at a cell density of 0.5 × 104/well and treated with fourfold serially diluted drugs for 72 h. Then 10 μl (5 mg/ml) of MTT salt (Sigma) was added to the corresponding wells. The cells were incubated at 37 °C for another 4 h, and the reaction was stopped by lysing the cells with 150 μl of DMSO for 5 min. The optical density was measured at 570 nm. A group that received no drug treatment was used as a reference for calculating the relative cell survival rate.
RNA analysis
Total RNA was isolated from cells during the logarithmic phase using TRIzol (Tiangen Biotech). For mRNA analysis, a cDNA primed by an oligo-dT was constructed using a PrimeScript RT reagent kit (Tiangen Biotech). The PON3 mRNA level was quantified using duplex-qRT-PCR analysis, wherein TaqMan probes with a different fluorescence profile were used to detect β-actin (provided by Shing Gene, Shanghai, China) in a FTC-3000P PCR instrument (Funglyn Biotech). Using the 2−ΔΔCt method, target gene expression levels were normalized to the β-actin expression level before the relative levels of the target genes were compared.
Western blot protein analysis
Cells were lysed with lysis buffer (60 mM Tris–HCl [pH 6.8], 2% SDS, 20% glycerol, 0.25% bromophenol blue, and 1.25% 2-mercaptoethanol) and heated at 95 °C for 10 min before electrophoresis/Western blot analysis. The primary anti-PON3 (17422-1-AP) antibodies and anti-GAPDH (60004-1-lg) antibodies were purchased from Proteintech (San Ying Biotechnology, China) and were recognized with anti-rabbit IgG peroxidase-conjugated antibody (30000-0-AP) (San Ying Biotechnology, China), followed by an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Waltham, MA, USA). Relative levels of proteins were quantified using densitometry with a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA). The target bands over the GAPDH band were densitometrically quantified, as indicated under each band (Additional file 1).
Wound-healing assays
For cell motility assays, cells stably expressing si-PON3, GFP-PON3 and the corresponding NC were seeded in 24-well plates and cultured to near confluence. After 6 h of culture in RPMI1640 without FBS, a linear wound was carefully made using a sterile 10 µl pipette tip across the confluent cell monolayer, and the cell debris was removed by washing with phosphate-buffered saline. The cells were incubated in RPMI1640 plus 10% FBS, and the wounded monolayers were then photographed at 0, 8, 12 and 20 h after wounding.
In vitro invasion assays
Cell invasion assays were performed in a 24-well plate with 8 mm pore size chamber inserts (Corning, USA). For invasion assays, 1 × 103 cells stably expressing si-PON3, GFP-PON3 or NC were placed into the upper chamber in each well with the matrigel-coated membrane, which was diluted in serum-free culture medium. In the assay, cells were suspended in 100 µl of RPMI1640 without FBS when they were seeded into the upper chamber. In the lower chamber, 500 µl of RPMI1640 supplemented with 10% FBS was added. After incubation for 36 h at 37 °C and 5% CO2, the membrane inserts were removed from the plate, and non-invading cells were removed with cotton swab from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were stained with 0.1% crystal violet for 30 min. The cells were then imaged and counted in at least 5 random fields using a CKX41 inverted microscope (Olympus, Japan). The assays were conducted in three independent times.
Signaling pathway analysis
The reporter construct encodes the firefly luciferase reporter gene under the control of a basal promoter element (TATA box) joined to tandem repeats of a specific transcriptional response element. The cells were transfected in triplicate with each firefly luciferase reporter construct in combination with the Renilla luciferase-based control construct using the riboFECT CP transfection reagent, and both the luciferase activities were measured in the cell extracts 24 h after transfection. The luciferase activities (luciferase unit) of the pathway reporter relative to those of the negative control in the transfected cells were calculated as a measurement of the pathway activity.
In vivo studies
Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male BALB/c nude mice between 3 and 4 weeks old were used for this study [14]. K510 cells were embedded in BD Matrigel Matrix (Becton, USA) and subcutaneously injected into two sites on the back of each mouse as follows: 1.0 × 107 cells/site for K510 into 2 sites/mouse, with 6 mice. Ten days after cell injection, all of the tumors were intratumorally injected with 2 nM NC/si-PON3 every 2 days. Ten days later, after four cell injections, three mice intraperitoneally received DDP (75 μg/mouse) once every other day. The remaining three mice in each group received PBS as a mock treatment control. The mice were euthanized on day 30 after four drug injections, and their tumors were weighed and imaged. Tumor weight was described as the mean ± S.D. The expression levels of PON3 and Ki67 proteins were measured using immunochemical analysis on 5 μm sections of formalin-fixed, paraffin-embedded tumor xenografts in nude mice. The antigens were retrieved by pre-treating the de-waxed sections in a microwave oven at 750 Watts for 5 min in citrate buffer (pH 6) processed with a Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy), and the slides were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. After the slides were counterstained with Mayer’s hematoxylin (Invitrogen), they were mounted in an aqueous mounting medium (Glycergel, Dako). Images were captured using a Leica DM 4000B microscope (Wetzlar, Germany), the relative level of each protein was calculated using Leica software (Wetzlar, Germany), and the percentage of the mock over the chemotherapeutically treated tumors was calculated and plotted.
Statistical analysis
All of the results are represented as the mean ± standard deviation (SD) of three independent experiments. Two-tailed Student’s t-test, one-way analysis of variance or Mann–Whitney U test was used to calculate statistical significance. All of the statistical analyses were performed with Microsoft Excel 2010 (Microsoft, Redmond, WA). A p-value of less than 0.05 was designated statistically significant.
