Patients and blood sample collection
All serum samples were collected from 98 neonates with sepsis and 50 neonates diagnosed with respiratory tract infection or pneumonia but without symptoms of sepsis inWeifang people's hospitalfrom 2013 to 2018. The neonatal sepsis patients who participated in the study were diagnosed based on clinical manifestations and blood pathogen detection according to the criteria that was established by the 2003 Kunming Neonatal Sepsis Definition Conference. The study has been approved by the Research Ethics Committee of the Weifang people's hospital, and all neonatal guardians are informed and provided paper-based informed letters.
RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)
Use Trizol reagent (Invitrogen, Carlsbad, CA, USA) to extract total RNA from the serum of neonates with sepsis and neonates with respiratory tract infection or pneumonia.PrimeScript RT reagent (TaKaRa, Shiga, Japan) uses RNA as a template for reverse transcription to obtain cDNA with the program of 42°C for 30min, 85°C for 10min.The serum levels of miR-141 and TLR4 mRNA were analyzed using SYBR-Green I Master Mix kit (Invitrogen, Carlsbad, California, USA) and 7300 Real-Time PCR system (Applied Biosystems, USA).U6 andGAPDH were respectively used as the internal control gene for miR-141 and TLR4. Three replicates were set up in this study. The final expression value was calculated using the 2-ΔΔCt method.
Cell culture and stimulation conditions
After adding 4.5% dextran to the blood sample of sepsis newborns and separating the white blood cells,mononuclear cells were isolated by density gradient centrifugation,and the purity of the cells was confirmed to be >95% by flow cytometry based on detection of the specific cell markers CD14 and CD45. The extracted mononuclear cells were cultured in RPMI-1640 medium containing 10% PBS at 37°C and 5% CO2. To explore the effect of MiR-141 on LPS-induced inflammation, monocytes were stimulated with 100ng/ml LPS for 4 hours.
The isolated monocytes were seeded on a 48-well plate, and the monocytes were transfected with MiR-141 mimic, MiR-141 inhibitor or negative controls (mimic NC and inhibitor NC; GenePharma, Shanghai, China) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to manufacturers’ protocols.
Luciferase reporter assay
Computer-aided algorithm (miRanda) was used to predict the target sequence of miR-141 in the 3'-UTR region of TLR4.To verify the relationship between MiR-141 and TLR4's 3'-UTR,a luciferase reporter assay was performed in this study.According to the predicted binding sites,the wild type (WT) and mutant (MT) TLR4 3'-UTR were ligated into pGL3 basic vector (Promega Corp.) to obtain the wild type (WT) vector pLUC-WT-TLR4 or mutant (MT) vector pLUC-MT-TLR4.miR-141 mimics, MiR-141 inhibitors or miR-NC were co-transfected into isolated monocytes with pLUC-WT-TLR4 or pLUC-MT-TLR4 using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.).The luciferase activity in different groups was measured using a dual luciferase reporter assay system (Promega Corp.).
The protein concentration of inflammatory cytokines IL-8 and tumor necrosis factor (TNF) -α in monocyte culture supernatant was evaluated using enzyme-linked immunosorbent adsorption co-precipitation (ELISA).This experiment was performed according to the instructions of the IL-8 ELISA kit (catalog number 550999; BD Biosciences) and the TNF-α ELISA kit (catalog number 550610; BD Biosciences).Finally, using the microplate reader (Bio-Rad Laboratories, Inc.) read the absorbance at 450nm.
All statistical analyses were performed by using SPSS 21.0 software and GraphPad Prism 5.0 software (GraphPad Software, Inc., USA). Values are expressed as the mean ± standard deviation and compared with Student's t-test, the χ2 test or one-way analysis of variance followed by Tukey's multiple-comparisons test. A receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic value of MiR-141 a regarding NS. P<0.05 was considered to indicate statistical significance.