2.1 Patients and samples
This descriptive study followed the recommendations of the ethics committee of the Changzhou Maternal and Child Health Care Hospital (approval number: 2021142) and was registered in the Chinese Clinical Trial Registry (approval number: ChiCTR2100049129). All participants and clinical data were collected from the Changzhou Maternal and Child Health Care Hospital from April to July 2021. BPD was defined as treatment with oxygen >21% for at least 28 days as proposed by the National Institute of Child Health and Human Development . The time point of assessment was 36-week postmenstrual age or discharge to home in infants with a GA <32 weeks, or >28 days but <56 days postnatal age, or discharge to home in infants with a GA >32 weeks, whichever came first . The inclusion criteria were as follows: preterm infants without genetic or structural anomalies, delivered at less than 32 weeks of gestation, and showing BPD (BPD group) or not (NBPD group). The exclusion criteria were as follows: pregnant women with infectious diseases; neonates with severe heart and lung malformations; and patients with severe hypoxic–ischemic encephalopathy, abnormal development of the intracranial hemorrhagic brain, or chromosomal abnormalities. Finally, eight UCB specimens were obtained from the umbilical vein right after fetal delivery (four BPD and four NBPD preterm infants). After clipping the umbilical cord, 5 mL of umbilical venous blood was immediately extracted from the placental end using a syringe and placed in a vacuum blood collection tube containing coagulant and inert separation glue. Then, the blood was laid aside at room temperature for 1 h. After the blood was curdled and the light yellow transparent liquid was precipitated, the collected samples were centrifuged at 1000g at room temperature for 10 min. Finally, the supernatant, which was umbilical venous blood serum, was extracted into new Eppendorf tubes and stored at –80°C until further use.
2.2 Isolation of exosomes from UCB serum
Exosomes were isolated following the protocol of ExoQuick exosome precipitation solution [Cat#EXOTC50A-1 (5 mL), System Biosciences (SBI), CA, USA]. First, 1 mL of UCB serum was centrifuged for 15 min at 3000g and 4℃ for the removal of cells and debris. The resulting serum was absorbed and added to 1.5-mL centrifuge tubes with 5 μL of thrombin (T4648-1KU, Sigma, MO, USA). After mixing, the samples were incubated for 15 min at 37℃. After centrifugation at 10,000g for 15 min at 4℃, the resulting supernatants were removed and the precipitated exosomes in the pellet were added with 250 μL of ExoQuick exosome precipitation solution. Then, specimens were mixed well and incubated for 30 min at 4℃. Exosomes were pelleted by 5-min centrifugation at 1500g at 4℃. The isolated exosomes were eluted in phosphate-buffered saline (PBS) and used immediately or stored at -80℃ for later use.
2.2 Nanoparticle tracking analysis
Exosome particle number was measured by nanoparticle tracking analysis (NTA) based on a previously published technique . In brief, exosomes diluted in PBS were analyzed by nanoparticle tracking using the ZetaView (Particle Metrix, Germany) equipment. A 405-nm excitation laser was used in instruments precalibrated with a 100-nm PSL standard (Applied Microspheres, Netherlands). NTA was performed with the same camera settings and tracking parameters, appropriate for detecting extracellular vesicle (EV) (sensitivity, 85; shutter, 70 min; brightness, 20 min; size, 10; maximum size, 200). Video acquisition was carried out at 30 frames/s, and videos were assessed for size and concentration using ZetaView.
2.3 Transmission electron microscopy
For transmission electron microscopy (TEM), exosomes pelleted by ultracentrifugation were resuspended in PBS. A drop thereof was placed on a copper mesh for 5 min. This was followed by 1-min staining with 1% phosphotungstic acid 44-hydrate and 20-min drying at room temperature. The preparations were examined under a transmission electron microscope (FEI, Tecnai G2 Spirit BioTwin; acceleration voltage, 80 kV).
2.5 RNA purification from exosomes and microarrays
Total RNA extraction uses an miRNeasy Serum Kit (Cat#217184, QIAGEN, GmBH, Germany) as directed by the manufacturer. RNA integrity was examined on an Agilent Bioanalyzer 2100 (Agilent Technologies, CA, USA). Then, total RNA amplification and labeling used a Low Input Quick Amp Labeling Kit, One-Color (Cat# 5190-2305, Agilent Technologies) according to the manufacturer’s protocol. Labeled circRNAs were obtained using an RNeasy Mini Kit (Cat.# 74106, QIAGEN, GmBH).
The slides were hybridized using 1.65 μg of Cy3-labeled circRNA and a Gene Expression Hybridization Kit (Cat. #5188-5242, Agilent Technologies) as directed by the manufacturer for 17 h. Staining dishes (Cat. # 121, Thermo Shandon, MA, USA) were used for washing with a Gene Expression Wash Buffer Kit (Cat.# 5188-5327, Agilent Technologies), according to the manufacturer’s protocol.
An Agilent Microarray Scanner (Cat. #G2565CA, Agilent Technologies) was used for scanning, with default settings. Data were extracted using Feature Extraction v10.7 (Agilent Technologies). Raw data were normalized using the Quantile algorithm and limma in R. Microarray analysis was carried out by Shanghai Biotechnology (China).
2.6 Functional enrichment analyses
Ratios were calculated between four preterm infants with BPD and four with NBPD. Genes showing fold changes ≥2 and P <0.05 (t test) were considered significantly differentially expressed. The chosen genes for exosomal circRNAs, lncRNAs, and mRNAs were analyzed using Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) with enrichment analysis software by Shanghai Biotechnology.
2.7 CircRNA/lncRNA–miRNA–mRNA network building
The miRanda database was used for predicting circRNA/microRNA (miRNA) interactions based on miRNA response elements (MREs) on circRNAs, with miRanda v3.3a. MREs on circRNA/lncRNAs were retrieved, and miRNAs were selected according to the seed matching sequences. For lncRNAs and mRNAs paired with the identical miRNA, the Pearson correlation coefficient (PCC) was determined for identifying the inferred circRNA/lncRNA–miRNA–mRNA pairs. Then, circRNA/lncRNA–miRNA–mRNA pairs showing PCC ≥0.90 were included to construct a circRNA/lncRNA–miRNA–mRNA network.
2.8 Cell culture and treatment
Human bronchial epithelial (BEAS-2B) cells and human umbilical vein endothelial cells (HUVECs) were provided by American Type Culture Collection (USA). These cells were routinely incubated in Dulbecco’s modified Eagle’s medium (Invitrogen, CA, USA) containing 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA) and 1% penicillin–streptomycin (Sigma–Aldrich, MO, USA) at 37ºC with 5% CO2 The BEAS-2B cells were treated with lipopolysaccharide (LPS, 1 µg/mL) for 12 h, and HUVECs were treated with LPS (1 µg/mL) for 18 h.
2.9 Cell counting kit-8 assay
For cell viability, the BEAS-2B cells and HUVECs were seeded in 96-well plates at a density of 1 × 104 cells/well stimulated with LPS (1 µg/mL) for 12 h and 18 h, respectively. Then, a cell counting kit-8 (CCK-8) (Beyotime Biotechnology, China) was used to examine the cell viability, according to the manufacturer’s specification. The optical density was detected at 490 nm using a microplate reader (Tecan Infinite M200 Micro Plate Reader; LabX, Switzerland).
2.10 Western blot analysis
Proteins extracted from BEAS-2B cells and HUVECs were measured using a bicinchoninic acid kit (Beyotime Biotechnology, China). Then, the proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The PVDF membranes were incubated using 5% skimmed milk, and then with primary antibodies at 4oC overnight. Blots were probed using the following antibodies: anti-IL-1β (1: 1, 000, ab234437; Abcam, Cambridge, UK), anti-TNF-α (1: 1, 000, ab183218; Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1: 2, 000, bs0755R; Bioss, China), with GAPDH being the endogenous control. Then, membranes were further incubated for 1 h using a secondary antibody (1: 2, 000, b-0311P-HRP; Bioss).
2.11 Quantitative real-time polymerase chain reaction
After extracting total RNA from LPS-induced BEAS-2B cells and HUVECs, cDNA was prepared with RNA using an RNeasy plus micro kit, as the starting material of quantitative polymerase chain reaction (qPCR), carried out using a Step One System (Life Technologies Corp). Subsequently, four differentially expressed circRNAs (hsa_circ_0086913, hsa_circ_0049170, hsa_circ_0087059, and hsa_circ_0065188) and two lncRNAs [small nucleolar RNA host gene 20 (SNHG20) and LINC00582] selected based on the P value and fold change were evaluated by quantitative reverse transcription (qRT)-PCR analysis. Primer Premier software 4.0 (Premier, Canada) was used to design sequences of all primers (see Table 3). GAPDH was normalized using the 2-ΔΔCTapproach.
2.12 Statistical analyses
SPSS 25.0 was used for data analysis. Quantitative data were expressed as mean ± standard deviation. Group pairs were compared using the Student t test. A P value <0.05 indicated statistical significance.