CircRNA circFADS2 is under-expressed in sepsis and protects lung cells from LPS-induced apoptosis by downregulating miR-133a | Journal of Inflammation

Sepsis patients and healthy controls

A total of 62 sepsis patients (male/female: 32/30; mean age, 52.3±4.9 years) who were admitted to Minhang Hospital, Fudan University between March 2018 and March 2020 were enrolled in the study. In addition, 62 healthy controls (male/female: 32/30; mean age, 52.4±4.8 years) who were at the Physiological Health Center of the hospital for routine systemic physical examination were recruited in the study. All healthy controls had normal physical parameters. To exclude other factors that could affect gene expression, patients with initiated therapy, other clinical disorders, and history of sepsis were excluded. All sepsis cases were caused by bacterial infections and diagnosed by blood test to show the existence of bacteria. All patients and controls signed informed consent. The current study was approved by the Ethics Committee of the Minhang Hospital, Fudan University. Blood samples were taken from sepsis patients within 24 h of admission. The blood samples of healthy participants were acquired during their physical examination.

Cell culture and LPS treatement

Blood samples (2 ml) were extracted from the elbow veins of both sepsis patients and healthy controls prior to therapy into tubes containing 0.2 ml of citric acids and centrifuged for 15 min at 1200 g to separate plasma.

Lung cells in this study were human bronchial epithelial cells (HBEpCs) from Sigma-Aldrich and cultured in bronchial Epithelial Cell Medium (Sigma-Aldrich) at 37 °C in an incubator with 5% CO2 incubator and 95% humidity to about 85% confluence prior to the subsequent assays. For LPS treatment, 1 × 106 HBEpCs were cultured in media containing 0, 2, 4, 8, and 12 µg/ml LPS for 48 h.

Knockdown assays

The small interfering RNAs (si-circFADS2 and si-NC) were synthesized and purchased from GenePharma (Shanghai, China). MiR-133a inhibitor and negative control (NC inhibitor) were purchased from Invitrogen. For knock-down assay, 1 × 106 HBEpCs were transfected with 50 nM siRNA or miR-133a inhibitor using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol.

Overexpression assays

CircFADS2 expression vector and miR-133a mimicwere purchased from GenePharma and Sigma-Aldrich (USA), respectively. For overexpression assay, 1 × 106 HBEpCs were transfected with 1 µg CircFADS2 expression vector or 50 nM miR-133a mimic using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Cells were washed with fresh medium after incubation with transfection medium for 6 h and cultured in fresh medium for 48 h prior to subsequent analysis.

RT-qPCR assays

Total RNAs in plasma and HBEpCs were extracted using Ribozol (VWR) and digested with a DNA eraser (Takara, Japan) until all samples reached an OD260/280 ratio close to 2.0, which indicated pure RNA. Electrophoresis (5% urea-PAGE gel) was carried out to analyze the integrity of RNA samples. Only RNA samples with high purity and satisfactory integrity were subjected to subsequent assays. The cDNA was synthesized using reverse transcription kit (Fermentas, USA). RT-qPCR assay was performed using ReverTra Ace™ qPCR RT Kit (Toyobo, Japan). GAPDH and U6 were selected as internal controls. Ct values of circFADS2 and miR-133a were normalized to their endogenous controls using the 2-ΔΔCt method. The primer sequences were presented in Supplementary Table 1.

Western blot

The whole cell proteins were extracted using RIPA lysis buffer (Gibco) and quantified by using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.).

After quantification, protein sample was separated by 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore Sigma, Billerica, MA). Afterward, the membrane was blocked in 5% non‑fat milk for 2 h at room temperature and incubated with anti-human cleaved caspase-3 (ab2302, 1:1,000, Abcam) and cleaved caspase-9 (ab2324, 1:1,000, Abcam) overnight at 4 ℃. After the incubation with goat anti-rabbit second antibody (ab205718, 1:5,000, Abcam), the protein levels were detected by enhanced chemiluminescence substrate (ECL, Millipore Sigma) and quantified using Image Lab™ Software (Bio-Rad).

MTT assay

Cells were grown in 96-well plates and underwent various treatments. On the next day, 10 µl of MTT (5 mg/ml, Sigma) was added to each well. After incubation for 4 h, cells were dissolved in 100 µl of dimethyl sulfoxide (DMSO, Sigma), and the absorbances at 570 nm were measured using a microplate reader (Molecular Devices, San Jose, CA) to determine cell viability.

Cell apoptosis assay

The apoptosis ratio was analyzed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China). In brief, HBEpCs were collected and transferred to 6-well cell culture plates with 8000 cells in 1.5 ml medium per well. After treatment, cells were digested with 0.25% trypsin-EDTA solution and then suspended by PBS. After being centrifuged at 1000 rpm for 5 min, 1 × 105 cells were incubated with 5µL Annexin V-FITC (BD Biosciences, USA) for 15 min and 5µL propidium iodide for another 5 min. Apoptotic cells were detected by flow cytometer (BD Biosciences, USA). Data were analyzed using CellQuest analysis software (BD Biosciences, USA).

ELISA

After different treatments, cell culture supernatants were collected, and the contents of interleukins tumor necrosis factor (TNF)-α (ab181421), IL-6 (ab178013), IL-8 (ab46032), and (IL)-1β were analyzed using ELISA kits (Abcam, Cambridge, UK) following the manufacturer’s instructions.

Dual‑luciferase reporter assay

Dual‑luciferase reporter assay was performed to detect the interaction between circFADS2 and miR-133a. circFADS2 wild-type (circFADS2-WT) or circFADS2 mutated type (circFADS2-Mut) reporter vectors were synthesized by General Biosystems. 1 × 106 HBEpCs were co‑transfected with circFADS2-WT (or circFADS2-Mut) and miR-133a (NC miRNA) using lipofectamine 3000. 48 h later, the luciferase activity was detected using the dual-lucy assay kit (Beijing Solarbio Science & Technology co., ltd.).

Statistical analysis

Data were presented as the mean ± standard deviation. Comparisons were conducted with a Student’s t test (for 2 groups) or one-way ANVOA followed by a Tukey’s post hoc test (for ≥3 groups). Pearson’s correlation coefficient analysis was performed to analyze the correlations between circFADS2 and miR-133a. A p<0.05 value was considered statistically significant. Experiments were repeated three times independently.

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