2.1. Animal protocols
Male C57BL/6J mice (3–4 weeks old) were supplied by Experimental Animal Center of Peking University Health Science Center. All animal care and experimental procedures in this study were approved by Animal Experimentation Ethics Committee of Peking University Health Science Center and complied with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication, 8th Edition, 2011). The C57BL/6J mice were fed with 60 kcal% high-fat diet (Research Diets, New Brunswick, NJ, United States) for four months to induce obesity and some obese mice were administrated with sitagliptin (3 mg/kg/day, Sigma-Aldrich Chemical, St Louis, MO, United States) for the last one month. Sitagliptin was dissolved in distilled water.
2.2. Measurement of GLP-1 in plasma
Mice were individually placed in a chamber containing room air and gradually introduced 100% CO2 at a displacement rate of 30–70% of the chamber volume per minute. CO2 flow was maintained for at least one minute after respiratory arrest. Then plasma was collected and kept. GLP-1 levels in plasma were examined by Glucagon-Like Peptide-1 ELISA kit (Linco Research, Inc, United States) according to the manual.
2.3. Detection of blood lipids
Levels of total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in plasma were detected by total Cholesterol Microplate Assay Kit (Signalway Antibody LLC, Shanghai, China), HDL Assay Kit (Langton Bio-tech, Shanghai, China), and LDL ELISA Kit (Langton Bio-tech, Shanghai, China) according to the manufacturers' instruction.
2.4. RNA extraction and lncRNA microarray
Total RNA was extracted from the fresh mouse aortae using TRizol reagent (Invitrogen, CA, USA). The RNA concentration and quality were determined using a NanoDrop ND-1000 (Thermo, MA, USA). RNA integrity was estimated by the standard denatured agarose gel electrophoresis. Microarray analysis was manipulated by KangChen Bio-tech (Shanghai, China). The Arraystar Mouse LncRNA Microarray Version 3.0 was utilized in this experiment. In accordance with the manufacturer's protocol, sample preparation and microarray experiments including labeling and hybridization were then accomplished.
2.5. Microarray data analysis
Agilent Feature Extraction software (Version 11.0.1.1) was applied to analyze the acquired array images. Quantile normalization and succedent data processing was manipulated by the GeneSpring GX v12.1 software package (Agilent Technologies). Differentially expressed (DE)-lncRNAs were selected with a fold change > 2 and P value < 0.05. DE-lncRNAs was identified by Volcano plot filtering and the expression patterns of DE-lncRNAs was discriminated by Hierarchical Clustering. The relevant sequence data in this study have been submitted to the NCBI GenBank databases under accession number GSE200814 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE200814).
2.6. Aortic ring preparation and functional assay
Mice were sacrificed by CO2 suffocation and then the thoracic aortae were removed and cut into ring segments, ~ 2 mm in length. Aortic rings were suspended in a wire myograph (Danish Myo Technology, Denmark) for detecting endothelium-dependent relaxations to acetylcholine (ACh, 10− 8 to 10− 5 mol/L) and endothelium-independent relaxation to sodium nitroprusside (SNP, 10− 9 to 10− 5 mol/L) as previously reported [18].
2.7. Western blot analysis
Protein extraction and Western blot were performed as previous described [18]. Antibodies against phospho-eNOS (Ser1177, 1:1000), eNOS (1:1000), phospho-AMPKα (Thr172, 1:1000), and AMPKα (1:1000) were purchased from Cell Signaling Technology (Beverly, MA, United States). Antibodies against Creb5 (1:500) was obtained from Sigma-Aldrich Chemical (St Louis, MO, United States). Antibody against β-actin (1:5000) was from Biodragon Technology (Beijing, China).
2.8. Primary culture of mouse aortic endothelial cells (MAECs)
Mice were were sacrificed by CO2 suffocation. The mouse aortae were placed in DMEM, cleaned of adhering tissues, and cut along the longitudinal axis. Then, the digestion and culture for cells were proceeded as previous described [18]. The endothelial cells were maintained until ~ 80% confluence.
2.9. Quantitative real-time PCR validation
By using NovoScript® 1st Strand cDNA Synthesis Super Mix (Novoprotein, Shanghai, China), RNA from mouse aortae or endothelial cells was reversely transcribed into cDNA. With the application of SYBR® qPCR Super Mix (Novoprotein, Shanghai, China), Quantitative real time polymerase chain reaction (qRT-PCR) was implemented by Thermo Fisher Piko real-time PCR system. All the experiments were carried out at least 3 times. The lncRNA and mRNA expressions were evaluated by the 2−ΔΔCt method and the calculation was then normalized to GAPDH expression. All primers were listed in supplementary Table S1 and Table S2.
2.10. Transfection condition
Small interfering RNA (siRNA) targeting lncRNA ENSMUST00000213271, Creb5, and negative control (NC) were obtained from HanBio (Shanghai, China), shown in supplementary Table S3. Aortic endothelial cells from Vehicle or obese mice were seeded in 12-well plates at ~ 70% confluence and transfected with respective siRNA (50 nmol/L) or corresponding NC by using Lipofectamine RNAiMax reagent (Invitrogen, CA, United States). Then, 12 hours later, the medium was changed. Cells were then collected for Quantitative real-time PCR or Western blot, 48 hours after transfection.
2.11. Statistical analysis
Results were represented as mean ± SEM. The relaxations were expressed as percentage of phenylephrine (Phe)-induced contraction. Data were analyzed using GraphPad Prism software (Version 8.0, San Diego, CA, USA). The protein expression was quantified by densitometer (FluorChem, Alpha Innotech, San Leandro, CA, USA) and analyzed by Quantity One software (Bio-Rad). Statistical significance was determined by two-tailed Student’s t-test and nonparametric test or one-way ANOVA followed by the Bonferroni post-hoc test when more than two treatments were compared. Two-way ANOVA was also applied in analyzing for the relaxations. P < 0.05 was considered statistically significant.