Animals
Male Balb/C mice (25-29g) were purchased from the Experimental Animal Center of Naval Military Medical University. The mice were housed in a temperature-controlled room with a 12-h light/dark cycle and access to food and water. All animal experiments were carried out according to the institutional criteria for the care and use of laboratory animals in research. The Shanghai Changhai Hospital Ethics Committee has reviewed and approved the study (the approval number is CHEC2019-008).
Animal model of sepsis
A total of 54 mice were randomly assigned into eight groups (Additional File 1: Figure S1): (a) sham operation group (n = 18), (b) CLP group (n = 18), (c) CLP plus si-control group (n = 3), (d) CLP plus si-NEAT2 group (n = 3), (e) CLP plus mimic control group (n = 3), (f) CLP plus miR-320 group (n = 3), (g) CLP plus normal saline group (n = 3), and (h) normal control group (n = 3). In group a and b, every 3 mice were divided into a subgroup according to six time points: 24 hours before the operation (T0), 24 hours after the operation (T1), 48 hours after the operation (T2), 72 hours after the operation (T3), 96 hours after the operation (T4), and 144 hours after the operation (T5). All mice were acclimatized for 7 days before the experiments.
The mice were anesthetized by an intraperitoneal injection of 10% chloral hydrate (4 ml/kg; Sangon, Shanghai, China) and fixed in a supine position. A midline incision (about 1 cm) was made on the anterior abdomen, and then the caecum was exposed and separated. For mice in the sepsis group, the cecum was exposed and ligated at the distal 30%. The cecum was then punctured twice with a sterile 18-gauge needle to extrude the fecal material. The cecum was placed back to the abdominal cavity and the incision was closed layer by layer. Mice in the sham group were treated with similar procedures but without ligation or puncture. The entire operation was carried out under aseptic conditions. After operation, all mice were resuscitated by injecting pre-warmed normal saline (37℃, 50 ml/kg) subcutaneously. Mice in group (c) to (g) were injected with lentivirus to knockdown NEAT2 and overexpression miR-320. Mice were returned to cages immediately at the end of the surgical procedures where access to water and food was available.
The mice in group (a) and (b) were sacrificed at the fixed time point, and in group (c) to (g) were sacrificed at 48 hours after the operation. Mice in group (h) were normal control group and were also used to culture normal EPCs for in vitro experiments. Blood samples and femurs were collected for the following experiments.
Peripheral cytokine and hepatorenal function assessments
In group (a) and (b), peripheral blood cytokines and hepatorenal function were assayed at the fixed time point, while in group (c) to (g) at 48 hours after the operation. The levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST) and creatinine (Cr) were detected using enzyme-linked immunosorbent assay (ELISA).
Circulation EPCs detection by flow cytometry
One hundred microliters of peripheral blood in mice were treated with 2ml red blood cell lysis buffer for 30 min at 0℃ and then were incubated for 30 minutes in the dark with P-phycoerythrin -labeled anti- CD309 antibody (BD Biosciences, San Jose, CA, USA) and fluorescein isothiocyanate- labeled anti-CD34 antibody (BD Biosciences, San Jose, CA, USA).
The analysis was performed using a FACSCalibur flow cytometer with CellQuest software (BD Pharmingen, San Diego, California, USA). The cell surface expression of CD309 was determined using 488/670 nm excitation and emission wavelengths. The cell surface expression of CD34 was determined by flow kilometric analysis using 490 to 494 nm wavelength laser excitations and monitoring the emitted fluorescence with a detector optimized to collect peak emissions at 520 to 525 nm.
Cell isolation and culture
Bone marrow from group (a) to (g) were collected for further experiments and from group (h) was used for culturing primary EPCs. Mononuclear cells (MNCs) were isolated from the bone marrow by density-gradient centrifugation with Ficoll (Sigma, Ronkonkoma, NY, USA). Immediately after isolation, MNCs (1×106/cm2) were plated in six-well culture dishes coated with 20 µg/ml of fibronectin (Chemicon, Temecula, CA, USA) and were maintained in endothelial growth medium-2 (EGM-2) (Lonza, Basel, Switzerland) supplemented with 20% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) for 2 hours. After 2 hours, in group (a) to (g), nonadherent cells were collected for PCR assay described below, and in group (h), nonadherent cells were collected and replanted. After 4 days in culture, nonadherent cells were removed by a thorough washing with PBS. The culture was maintained through day 7, and adherent cells were subjected to further examinations.
Characterization of endothelial progenitor cells
To confirm the phenotype, immunofluorescence analysis and fluorescent chemical detection of EPCs were performed on adherent mononuclear cells after 7 days in culture. Adherent mononuclear cells were fixed with 4% paraformaldehyde for 15 min and permeated in PBS containing 0.5% Triton X-100 (Beyotime Biotechnology, Shanghai, China) for 20 min at room temperature followed by incubation in 5% BSA blocking buffer. The primary antibodies complexes were visualized using P-phycoerythrin-labeled anti-CD 31 (BD, New York, USA) and P-phycoerythrin-labeled anti-CD 309 (BD, New York, USA). Nuclei were counterstained with 0.5 µg/ml of 4', 6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology, Shanghai, China).
Additionally, direct fluorescent staining was used to detect dual binding of FITC-labeled Ulex europaeus agglutinin (UEA-1) (Sigma-Aldrich, St. Louis, MO, USA) and 1,1-dioctadecyl- 3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-ac-LDL) (Molecular Probe, Carlsbad, California, USA). Cells were first incubated with Dil-ac-LDL at 37°C and later fixed with 2% paraformaldehyde for 10 minutes. After being washed twice, cells were reacted with UEA-1 (10 mg/l) for 1 hour. After the staining, samples were analyzed using a laser scanning confocal microscope (Olympus, Tokyo, Japan).
Cell transfection
The lentiviral sgRNA against the lncRNA NEAT-2 vector and siRNA oligonucleotides were synthesized by Genechem company (Shanghai, China). EPCs were seeded at 3×105cell/well in a 6-well plate and pre-incubated in the cultured medium without antibiotics for overnight, until 50% confluence. Then, cells were transfected with vector or siRNAs (50 nM) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA)) following the manufacturer’s protocol.
Luciferase reporter assay
The miR-320 binding site of NEAT2 was predicted using the bioinformatics databases (Starbase v2.0, miRcode and RNAhybrid). The fragment from 3’UTR of NEAT-2 containing the putative binding site of miR-320 was synthesized and subcloned into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to form the reporter vector NEAT2-wild-type (NEAT2-Wt) (Genechem, Shanghai, China). To test the binding specificity, the mutant of putative miR-320 binding sites was created to form the reporter vector NEAT2-mutated-type (NEAT2-Mut) (Genechem, Shanghai, China). Then miR-320 mimic or negative control mimic was co-transfected with the reporter vector containing the corresponding mutants using Lipofectamine 2000®, according to the manufacturer’s instructions. Luciferase reporter assay was performed according to the manufacturer’s instructions.
Quantitative real-time PCR analysis
After the indicated treatments, total RNA was extracted from nonadherent MNCs using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then subjected to reverse transcription-PCR with ImProm-II™ (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Real-time PCR analysis for NEAT-2 expression was performed on a LightCycler thermocycler (Roche, Basel, Switzerland) by using SYBR Master Mixture (TaKaRa, Tokyo, Japan).
The Taqman MicroRNA Reverse Transcription Kit and Taqman Universal Master Mix II with the TaqMan MicroRNA Assay of miR-320 and U6 were used for testing the expression levels of miR-320. Primers used here were as follows: NEAT-2, F: 5’- CAGACCACCACAGGTTTACAG-3’ and R: 5’- AGACCATCCCAAAATGCTTCA-3’; GAPDH, F: 5’- TGACTTCAACAGCGACACCCA-3’ and R: 5’- CACCCTGTTGCTGTAGCCAAA-3’. MiR-320 was normalized to U6, and NEAT2 was normalized to GAPDH expression.
3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, migration assay and angiogenesis assay
EPCs were cultured in 96-well plates (10,000/well) for 3 days prior to the addition of MTT. These plates were further incubated for 4 h; subsequently, dimethyl sulfoxide (DMSO; 160 µL) was put into each of those wells and the plates were vortexed for 8 min. The optical density (OD) at 490 nm was measured using a micro-plate reader. The growth chart was mapped with OD on the Y axis and time interval as the X axis. The cell viability was subsequently detected using the MTT method.
Cell migration was measured by using transwell chambers (Corning Co., Corning, NY, USA). Briefly, EPCs were re-suspended in Opti-MEM® I reduced serum medium and 2×104 EPCs were seeded on the upper chamber of 24-well with the volume of 200 µl. At the same time, 600 µl of EGM-2 medium was added to the lower chamber. After 24 h of incubation at 37°C in a humidified atmosphere of 5% CO2 cells were fixed in 4% paraformaldehyde and non-traversed cells remaining in the upper membrane were scraped off cotton swab. Traversed cells in the lower side of the membrane were stained with 0.1% crystal violet. Under optical microscope, traversed cells were counted from five randomly chosen fields at 100× magnification and the results were reported as the number of cells per field.
Briefly, EPCs (5 × 104 cells) and EPCs infected with si-control, NEAT2 siRNA, miR-320 mimic, mimic control lentivirus were seeded in Matrigel-coated 48-well plates, after which the tubular structures of EPCs in the Matrigel were examined under a microscope after 12 hours of incubation. The total number of tubes that formed, which represents the degree of angiogenesis in vitro, was scanned and quantified in three random fields (×100 magnification) per well.
Statistical analysis
All data were analyzed using the SPSS 23.0 statistical package (SPSS Inc., Chicago, IL, USA). The significance level for all of the statistical tests was set at 0.05. All statistical tests were two-sided. Continuous values were analyzed with mean and standard deviation. Student’s t test was applied to compare continuous variables, and chi-square test (or Fisher’s exact test in specific condition) was applied to analyze categorical variables when comparing differences among various groups.