Materials and reagents
Bpv (PTEN) and 5-Aza-2′-deoxycytidine (5-Aza) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Complete Freund’s adjuvant was obtained from Chondrex, Inc. (Redmond, WA). Rabbit anti-PTEN, anti-TIMP metallopeptidase inhibitor 1 (TIMP-1), anti-TNF-α antibody and mouse anti-DNA methyltransferase 1(DNMT1) monoclonal antibody were purchased from Abcam (Cambridge, UK). Rabbit anti-IL-1β, anti-IL-6 and anti-IL-17A, and mouse anti-β-actin monoclonal antibody were obtained from Bioworld (Shanghai, China). Rabbit anti-MMP-3, anti-MMP-9, anti-AKT and anti-p-AKT antibody, and Simple ChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) were purchased from Cell Signaling Technology (Danvers, MA). Peroxidase-Conjugated Goat anti-Rabbit IgG (H+L) were purchased from ZSGB-BIO (Beijing, China). The primers of PTEN, IL-1β, IL-6, IL-17A, IL-8, IL-10, MMP-3, MMP-9, TIMP-1, CCL-2, CCL-3, CCL-8 and β-actin were synthesized by Sangon Biotech Co., Ltd (Shanghai, China).
Human synovium and FLS
Human synovium or FLS were extracted from patients with RA (6 patients) or OA (8 patients) undergoing total joint replacement. All patients were collected at the Department of Orthopedics, First Affiliated Hospital, Anhui Medical University, Hefei, China. And all patients with RA met the American College of Rheumatology 1987 revised criteria for seropositive RA as previously described. FLS were used between p4 and p9 passages.
Adjuvant-induced arthritis (AIA) of rat model
The AIA model was induced by Sprague-Dawley rats (80-120 g, female) were treated with Complete Freund’s adjuvant (Chondrex, Inc, 0.1 mL/100 g body weight) for 24 days by subcutaneously injection in the left hind paw [7, 21]. At same time, normal control rats were injected with normal saline. After 7 days, AIA rats were treatment with adenovirus carrying rattus PTEN (ad-PTEN) and 5-Aza. The 0.1 mL ad-PTEN or ad-GFP was intraarticular injected into AIA hind knees. And 5-Aza was intraperitoneal injected at a dose of 0.7 mg/kg/3 days for 21 days. The rats were provided from the Experimental Animal Center of Anhui Medical University. And efforts were made to reduce the number of animals used and their suffering in all animal experiments. All experimental protocols used on the animals were approved by the institutions’ subcommittees on animal care of Anhui Medical University (approval number: 20160253).
The synovium specimens from human and rats’ knee joint were fixed by 4% paraformaldehyde for 48 h and embedded by paraffin. According to a standard procedure, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and immunofluorescence (IF) were performed. And the pathological changes were assessed and photographed under CaseViewer (3DHISTECH Ltd., Hungary).
Enzyme-Linked Immunosorbent Assay (ELISA) assay
After AIA treatment with ad-PTEN and 5-Aza for 21 days, the serum of rats was collected through abdominal aorta. And the RA FLS were treated for 48 h, the supernatant was collected by centrifugation. The levels of Rat IL-6 and TNF-α, human IL-6 and IL-8 were determined by ELISA kit (R&D, Minneapolis, USA) according to the manufacturer’s protocol.
Isolation of peritoneal macrophages
Peritoneal macrophages were collected from the peritoneum of AIA by douched with PBS. Cells were placed in 6 well plates at 2.0-5.0 × 106 with high-glucose DMEM supplemented with 10 % (v/v) FBS (PAN Biotech, Germany). Adherent cells were harvested from the plates after 2 h of culture.
Isolation of peripheral blood mononuclear cell (PBMC)
The blood was collected from rats AIA through abdominal aorta. And the samples were prepared with human peripheral blood lymphocyte separation fluid (Tianjin Hao Yang, China) according to the manufacturer’s protocol prior to RNA extraction and protein analysis.
FLS were derived by tissue direct separation method from rats AIA and human RA synovial and cultured in DMEM (HyClone, South Logan, UT, USA) supplemented with 20 % (v/v) FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Beyotime, Shanghai, China) at 37 °C and in an atmosphere of 5 % CO2.
The FLS were plated at a density of 1.0-2.0×105 cells/ml in 6-well plates for 24 h. After FLS treatment with TNF-α, immunocytochemistry staining was performed with rabbit anti-PTEN. And the Alexa Fluor 488-Conjugated Goat anti-rabbit IgG (H+L) (ZSGB-BIO, Beijing, China) and 4', 6-diamidino-2-phenylindole (DAPI; Beyotime, China) were incubated in dark. And then the cells were photographed under an Olympus BX-51 microscope (Olympus, Tokyo, Japan).
Small interfering RNA silencing and plasmid construction
According to the manufacturer’s instructions, FLS were transfected with small interfering RNA (RNAi; GenePharma, Shanghai, China) or over-expression plasmid (GeneChem, Shanghai) using lipofectamine TM 2000 (Invitrogen, Carlsbad, CA). The oligonucleotide sequences were as follows: PTEN-RNAi (rat), 5'-CCGAUACUUCUCUCCAAAUTT-3' for the sense strand and 5'-AUUUGGAGAGAAGUAUCGGTT-3' for the antisense strand; PTEN-RNAi (human), 5'-CAGUAGAGGAGCCGUCAAATT-3' for the sense strand and 5'- UUUGACGGCUCCUCUACUGTT-3' for the antisense strand. A negative scrambled RNAi was used in parallel. And the FLS were transfected with rat PTEN-GV141 (rat) and PTEN-pcDNA3.1 (human) to induce the over-expression of PTEN, and with empty GV141 vector (GV141) or empty pcDNA3.1 vector as control. After transfection for 8 h, the FLS were cultured with complete medium at 37 °C for 48 h.
For recombinant adenovirus construction, over-expression of PTEN adenovirus (Ad-PTEN) and the negative control adenovirus (Ad-GFP) were obtained from Hanbio (Shanghai). The stock solutions of Ad-PTEN and Ad-GFP were 1×1010 PFU/mL, respectively.
Methylation-specific PCR (MSP)
DNA samples treated with a Wizard® DNA Clean-Up System (Promega, Madison, WI) according to the manufacturer’s instructions. Unmethylated cytosine residues in the DNA samples were converted to uracil using a Methylamp™ DNA Modification Kit (EpiGentek, Farmingdale, NY). Primers of methylated and unmethylated PTEN were as follows: PTEN (human, methylated) forward: 5'-GATGAGGTGATATACGTTGGCG-3' and reverse: 5'-TTTACACCGCTATCGAATCACAAT-3'; PTEN (human, unmethylated) forward: 5'-GGATGAGGTGATATATGTTGGTGAT-3' and reverse: 5'-TTTTACACCACTATCAAATCACAATCA-3'; PTEN (rat, methylated) forward: 5'-CGGTCGGTGTTAAGTTTTTCGT-3' and reverse: 5'-AAAACGAATAATCCTCGCAACG-3'; PTEN (rat, unmethylated) forward: 5'-ATTTGGTTGGTGTTAAGTTTTTTGT-3' and reverse: 5'-AAAAAACAAATAATCCTCACAACAAAC-3'.
Quantitative real-time PCR (q-PCR)
Total RNA was extracted by classical TRIzol reagent (Invitrogen) from FLS and reverse transcribed to cDNA with iScriptTM cDNA kit (Bio-Rad, CA). Then, the q-PCR reaction mixture comprises cDNA and SYBR Green q-PCR Master Mix (TOYOBO, Japan). Primer sequences are shown in Supplementary Table 1. All reaction was conducted 3 times, and the relative mRNA expression of target genes was obtained by normalization to the levels of β-actin.
The total protein was extracted by lysis buffer from FLS and denaturted by boiling. Then, cell extract was isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blot onto PVDF membranes (Millipore, Bedford, MA). After blocking, it was incubated with primary antibody overnight. Rabbit antibodies (PTEN, IL-1β, IL-6, IL-17A, MMP-3, MMP-9 and TIMP-1) were used at a dilution of 1:500, and mouse anti-DNMT1 and anti-β-actin were used at a dilution of 1:1, 000. Then, after washing, the blot was incubated with goat anti-mouse or anti-rabbit HRP-conjugated antibodies for 1 h. The protein blot bands were photographed by ChemiDocTM MP Imaging System (Bio-Rad) with ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore).
FLS were cultured in 24-well plate (5.0× 105/mL cells/ well). After cells reached to 60%-80%, the FLS treated with bpv, PTEN-RNAi or PTEN-GV141 for 24 h. And then, cells were serum deprived and scratched with a pipette tip. After 48 h and the cells were fixed with methanol, stained with crystal violet, and photographed by Olympus BX-51 microscope (Olympus).
Chromatin Immunoprecipitation (ChIP)
The SimpleChIP® Kit (Cell Signaling Technology) was performed according to manufacturer's instructions. After FLS were cross-linked in 1% formaldehyde and lysed, it was immunoprecipitated using anti-DNMT1 monoclonal antibody overnight at 4 °C, and incubated with ChIP-grade Protein A/G Plus agarose beads for 2 h. After washing with three different buffers, the samples reverse cross-linked at 65 °C for 2 h, and detected ChIP signals by q-PCR.
Data are presented as the means ± standard deviation and analyzed using SPSS16.0 software. Statistical significances were determined by one-way ANOVA with a post-hoc Dunnett’s test. In all cases, values of P<0.05 were considered to be statistically significant.