SET8 suppression mediates high glucose-induced vascular endothelial inflammation via the upregulation of PTEN

SET8 suppression mediates high glucose-induced vascular endothelial inflammation via the upregulation of PTEN

ParticipantsThere were 50 newly diagnosed patients with type 2 diabetes mellitus (T2DM) and 30 healthy volunteers included in the present study. This study conformed to the Declaration of Helsinki and was approved by the Ethics Committee of Huzhou Central Hospital (20191209-01). Written informed consent was acquired from each participant prior to enrollment. The definition of T2DM included the following: fasting plasma glucose levels were ≥126 mg/dl, HbA1c levels were ≥6.5%, plasma glucose levels after 2 h were ≥199.8 mg/dl or a random plasma glucose level was ≥199.8 mg/dl. The exclusion criteria included advanced liver disease, renal failure, valvular heart disease, severe heart failure, stroke, atrial fibrillation, peripheral arterial disease, and other vascular diseases.Collection of venous blood samplesIn the present study, EDTA vacutainer tubes were used to collect fasting venous blood samples from all participants. The plasma samples were collected and then stored at −80 °C until analysis.Collection of peripheral blood mononuclear cellsIn the present study, Ficoll standard density gradient centrifugation was employed to collect peripheral blood mononuclear cells. The upper layer containing peripheral blood mononuclear cells was collected and then stored at −80 °C until analysis.Rat model of T2DMMale Sprague-Dawley rats weighing 200–300 g were employed in this study. The rats were acquired from Shanghai SLAC Laboratories. The present study complied with the Guide for the Care and Use of Laboratory Animals of Zhejiang University Laboratory Animal Welfare Ethics Review Committee and was performed according to the Institutional Guidelines for Animal Research and the Guide for the Care and Use of Laboratory Animals published by the US NIH (2011). Ten rats were randomly divided into the control group (con, n = 5) and the diabetic group (DM, n = 5). For the con group, the rats were injected intraperitoneally once with citrate buffer only (0.1 M, pH 4.5). For the DM group, after feeding the rats a high-sugar/high-fat diet (67% basic feed, 10% lard, 20% sugar, 2.5% cholesterol, and 0.5% sodium cholate) for 2 weeks, the rats received a single intraperitoneal injection of streptozotocin (STZ, 50 mg/kg) and were then moved back to a standard laboratory chow for 4 weeks. Hyperglycemia was verified by detecting blood glucose through tail-neck blood sampling one week after STZ injection.Collection of rat blood samplesRat blood samples were gathered by cardiac puncture. Euthanasia of all the rats was performed by intraperitoneal administration of thiopental sodium (40 mg/kg). Rat blood samples were collected in EDTA vacutainer tubes. The plasma samples were collected and then kept frozen at −80 °C until analysis.Detection of sICAM-1 and e-selectin levelsHuman and rat sICAM-1 and e-selectin were detected with enzyme-linked immunosorbent assay kits (Meimian Industrial Co., Ltd, Jiangsu, China).Cell cultureHUVECs (ATCC; Manassas, USA) were cultured in low-glucose (5 mM) Dulbecco’s modified Eagle’s medium (DMEM; HyClone Laboratories, Logan, USA) with 10% fetal bovine serum and 1% penicillin–streptomycin. The experimental group was cultured in high-glucose (25 mM) DMEM with 10% fetal bovine serum and 1% penicillin–streptomycin for 6 days. THP-1 cells (ATCC; Manassas, USA) were cultured in RPMI 1640 medium (HyClone Laboratories, Logan, USA) with 10% fetal bovine serum and 1% penicillin–streptomycin. The culture environment was created by an incubator containing 5% CO2 at 37 °C. The condition of 5 mM glucose plus 20 mM mannitol (Sigma, St. Louis, MO) was employed as an osmotic control in this study.Adhesion of mononuclear cells to HUVECsFirst, we cultured HUVECs in 5 mM glucose or 25 mM glucose DMEM for 6 days. Then, 30,000 THP-1 cells were added to the HUVECs and coincubated at 37 °C for 30 min. After that, the cells were washed with PBS three times and analyzed by phase-contrast microscopy. We counted five separate culture dishes in 10 microscopic fields of view.Western blot analysisProteins were extracted from the HUVECs of the different cultures by cell lysis buffer (Cell Signaling Technology, Danvers, MA). Equal amounts of protein were separated by 10% SDS–PAGE gels and then transferred to PVDF membranes (Millipore Corporation). The PVDF membranes were blocked in 5% skim milk at room temperature for 1 h and then incubated with the corresponding primary antibody (1:1000) at room temperature for 2 h. The primary antibodies were monoclonal antibodies against β-actin (ProteinTech, Wuhan, China), PTEN (ProteinTech, Wuhan, China), SET8 (ProteinTech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), forkhead box protein O1 (FOXO1) (Cell Signaling Technology, Danvers, MA), and e-selectin (Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal antibodies against ICAM-1 (Cell Signaling Technology, Danvers, MA) and p-p65 (Cell Signaling Technology, Danvers, MA). The membranes were incubated with the corresponding secondary antibody (1:1000) at room temperature for 1 h. The membranes were then washed, and the proteins were detected by a LAS-4000 mini CCD camera (GE Healthcare).RNA extraction and quantitative real-time PCR (qRT-PCR)Total RNA was obtained by TRIzol (Invitrogen, Grand Island, NY, USA). According to the manufacturer’s instructions, PrimeScript RT reagent (TaKaRa) was used to synthesize complementary DNA (cDNA). Then, we used Hieff UNICON® qPCR TaqMan Probe Master Mix (Yeasen, Shanghai, China) for qPCR on an ABI7500 Real-Time PCR system (Applied Biosystems). The primers used in the present study are shown in Supplementary Table 1.Immunohistochemistry (IHC)The animal tissues were embedded in paraffin and then processed for IHC. The paraffin sections were incubated with anti-SET8 (ProteinTech, Wuhan, China), anti-FOXO1 (Cell Signaling Technology, Danvers, MA), anti-PTEN (Proteintech, Wuhan, China), and anti-CD31 (Proteintech, Wuhan, China) antibodies overnight at 4 °C in a humidified chamber. Double staining was performed with the use of diaminobenzidine and fast red as the enzyme substrates according to the manufacturer’s instructions.siRNA, shRNA, and SET8 mutant treatmentsHUVECs were transfected with shSET8, a mutant SET8R295G plasmid15, siFOXO1 and siPTEN with the use of Lipofectamine 3000 (Invitrogen, USA).The sequences of shSET8 (Biotend, Shanghai, China) were shRNA-a, 5′-CAACAGAATCGCAAACTTA-3′, and shRNA-b, 5′-CAACAGAATCGCAAACTTA-3′. The sequences of siFOXO-1 (Biotend, Shanghai, China) were siRNA-a, 5′-CCCAGUCUGUCUGAGAUAATT-3′, and siRNA-b, 5′-UUAUCUCAGACAGACUGGGTT-3′. The sequences of siPTEN (Biotend, Shanghai, China) were siRNA-a, 5′-GGUGUAAUGAUAUGUGCAUdTdT-3′, and siRNA-b, 5′-CAAAUUUAAUUGCAGAGUUdTdT-3′.Coimmunoprecipitation (Co-IP)Protein extracts were prepared with the use of cell lysis buffer containing PMSF (Beyotime Biotechnology, Shanghai, China). A total of 30 μl of the cell extract was used as the input. For endogenous IP, the cell extracts were cultured with the corresponding primary antibodies and 50 μl of protein A/G Dynabeads (Thermo Fisher, USA) at 4 °C overnight. Then, 10 μl of the input, IgG, and IP extracts were subjected to western blot.Immunofluorescence (IF) stainingHUVECs with the corresponding treatment were seeded onto glass slides. After fixing with 4% paraformaldehyde, the cells were permeabilized with 0.3% Triton X-100 for 5 min and then blocked for 1 h at room temperature. The cells were incubated with anti-SET8 (Proteintech, Wuhan, China) and anti-FOXO1 (Cell Signaling Technology, Danvers, MA) antibodies overnight at 4 °C. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. The images were photographed with a confocal Leica fluorescence microscope.Chromatin immunoprecipitation (ChIP) assayChIP assays were performed with the use of a Simple ChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, MA). Briefly, the cells (1 × 107) were fixed with 1% formaldehyde for 10 min at room temperature to cross-link the DNA and the proteins. The cross-linking reaction was then stopped with the use of glycine. A Microson XL ultrasonic cell disruptor XL (Misonix) was used to shear the chromatin. Ten microliters of the sonicated solution was gathered as an input control. The surplus sonicated solution was incubated with anti-FOXO1 antibody (Cell Signaling Technology, Danvers, MA), anti-H4K20me1 (Abcam, Cambridge, UK) antibody or a negative control IgG at 4 °C overnight. The immunoprecipitants were bound to protein G magnetic beads, and the DNA–protein cross-linking was terminated by incubation at 65 °C for 2 h. After purification, the enriched DNA sequences were detected by PCR. To confirm whether FOXO1 binds to the methylated promoter of PTEN, a re-ChIP assay was performed. In brief, after the standard ChIP assay, the beads were incubated with 10 mM dithiothreitol for 30 min at 37 °C. The eluent was then diluted with sonication buffer, followed by a second round of the ChIP assay. The PTEN oligonucleotide primer sequences were forward, 5′-TTGGATGTGGGTGCTTGTGT-3′, and reverse, 5′-CTTCTTCCTTTGCTCGGGGT-3′.Dual-luciferase assayThe effect of SET8 and FOXO1 on the activity of the PTEN promoter was evaluated by a Promega Dual-luciferase Assay Kit (Madison, WI, USA). The PTEN promoter was amplified and ligated into the pGL3-basic vector to create the pGL3–PTEN construct. The pGL3–PTEN plasmid was transfected along with a Renilla luciferase vector into HUVECs. The effect of SET8 and FOXO1 on PTEN promoter activity was assessed using a dual-luciferase assay kit.Statistical analysisThe data were acquired from at least five experiments performed separately, and the results are shown as the mean ± SD (standard deviation). Two-tailed unpaired t-tests or one-way ANOVAs performed with GraphPad Prism Version 7.0 (GraphPad Software, San Diego, CA) were used to compare the groups. P < 0.05 was considered significant.

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