Human tissue specimens were collected from PA patients, including 20 cases of invasive and 20 cases of non-invasive pituitary tumor. Meanwhile, 20 cases of normal pituitary tissues were obtained from the thin layer of pituitary tissue around the ACTH microadenocarcinoma as control group. All samples were obtained from the Second Affiliated Hospital of Guilin Medical University (Guilin, Guangxi, China). After surgical resection, all the tissue samples were immediately frozen in liquid nitrogen and stored at –80 °C until further analysis. Before tissue collection, all patients were administered the informed consent and this study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Guilin Medical University.
Rat PA cell lines, including MMQ, GH1, GH3 and RC-4B/C, and normal pitultary cells were purchased from the American Type Cell Collection (ATCC, Manassas, VA, USA). MMQ, GH1 and GH3 were routinely cultured in ATCC-formulated F-12K medium (Invitrogen, Carlsbad, CA). RC-4B/C and normal pituitary cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Invitrogen). All the media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 units/mL penicillin, and 100 units/mL streptomycin. All cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37°C.
Cell treatment and transfection
GH3 cells were used for pharmacological demethylation. Briefly, approximately 1 × 106 cells were seeded in six-well plates and treated with 5-Azacytidine(5-AzaC, Sigma-Aldrich, St. Louis., MO, USA) for 24 at 37°C. For cell transfection, small interfering RNA targeting TIMP2 (siTIMP2), TIMP3 (siTIMP3) and control siRNA (siNC) were chemically synthesized by GenePharma Co. Ltd. (Shanghai, China) and transfected into GH3 cells with a final concentration of 10 nM. Coding sequence of human TIMP2 or TIMP3 was sub-cloned into pcDNA3.0 vector (Invitrogen) to construct TIMP2 or TIMP3 overexpression vector by RiboBio (Guangzhou, China). The plasmid vectors, including pcDNA3.0-TIMP2, pcDNA3.0-TIMP3 or pcDNA3.0 were transfected into the GH3 cells. All cell transfections were performed in accordance with the manual for the reagent transfection Lipofectamine 2000 (Invitrogen). After 48 h transfection, the cells were harvested for the subsequent experiments.
Quantitative real time PCR
Total RNA was extracted from tissue samples or cell lines using TRIzol reagent (Invitrogen) and reverse transcribed using M-MLV reverse transcriptase (Bio-Rad, Minneapolis, MN, USA)by extension of oligo primers (TaKaRa). Quantitative real time PCR was performed with DBI Bestar® SybrGreen qPCR Master Mix on a Stratagene Real time PCR (Mx3000P, Agilent). The mRNA expression was calculated by using the 2−ΔΔCt method and normalized to the expression of GAPDH mRNA. The primer sequences are shown in Table 1.
Western blot analysis
Total protein was extracted from tissue samples or cell lines using RIPA lysis buffer (Roche, Complete Mini). The protein concentration was determined using bicinchoninic acid method. Approximately 30 µg of the total protein was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting analysis and electro-transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories Inc., USA). After blocking with Tris-buffered saline containing 0.1% Tween 20 (TBST, Sigma, USA), the membranes were incubated with primary antibodies against TIMP1 (1:1000), TIMP2 (1:2000), TIMP3 (1:4000), DNMT1 (1:1000), DNMT3a (1:2000), DNMT3b (1:1500) and GAPDH (1:10000) all from Abcam (Cambridge, UK) at 4°C overnight. On the next day, the membranes were washed with TBST three times (5 min each time), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:20000, BA1051, BA1054) for 2 h at room temperature. Subsequently, the protein signals were detected using enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
Methylation-specific PCR (MSP)
Genomic DNA was extracted from tissues or cells using High Pure PCR Template Preparation Kit (Roche Applied Science, Pennsburg, Germany). The sequence of promoter 5′-C-phosphate-G-3′ (CpG) islands of the relevant genes was predicted online (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). The DNA samples were treated with sodium bisulfite using the EpiTect Bisulfite Kit (QiaGen, Hilden, Germany), followed byDNA modification by EZ DNA Methylation-Gold Kit (Zymo Research). Bisulfite DNA was amplified with methylated or unmethylated specific primer sequences designed by Methyl primer Express v1.0, as listed in Table 2. MSP was used to amplify methylated gene and MSP products were analyzed by 2% agarose gel electrophoresis. Methylated and non-methylated human DNAs were used as positive and negative controls, respectively.
Treated or transfected GH3 cells at a density of 3,000 cells per well were seeded in a 96-well plate and incubated overnight at 37°C. Then, cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan). Briefly, 10 μL CCK-8 solutions was added to each well at 24, 48, and 72 h, respectively, followed by incubation for 2 h at 37°C. The optical density values were measured using a microplate reader (Bio-Tek, USA) at 450 nm.
Wound healing assay
Wound healing assay was performed to evaluate cell migration ability. Briefly, GH3 cells at a density of 4, 0000 cells per well were seeded in six-well plates and cultured until 90% confluence. Subsequently, a 100-μL sterile pipette tip was used to artificially create a straight scratch through the monolayer. The images of the same location at 0 and 48 h after wounding in five randomly selected fields were observed under a microscope (Olympus, Tokyo, Japan). The relative average migration distance was calculated as follows: (Width0h − Width48h)/Width0h×100%.
Cell invasion assay
For cell invasion assay, approximately 3 × 105 cells in serum free medium were added to the upper chamber coated with Matrigel (Millipore) in 24-well transwell insert. Then, 500 μL serum-containing medium was added to the lower chamber. After 48 h incubation, cells that had invaded to the lower chamber were fixed and stained with 0.5 % crystal violet. Stained cells were photographed and counted in five randomly selected fields under a light microscope.
Quantitative data were analyzed with SPSS 21.0 software package and expressed as mean ± standard deviation (SD) of at least three repetitive experiments. Comparisons of continuous data were performed using two-tailed t-test for two groups or one-way analysis of variance for multiple comparisons data. The values of p less than 0.05 were considered to be statistically significant differences.