Sepsis patients and healthy controls
Research subjects in this study were both sepsis patients (n=60, 34 males and 26 females; 38 to 62 years; 49.8±5.6 year) and healthy controls (n=60, 34 males and 26 females; 38 to 62 years; 49.9±5.7 year). All the participants were enrolled at No.1 Hospital Attached to Jiamusi University from May 2017 to May 2019. All sepsis patients were diagnosed for the first time. The main cause of sepsis was bacterial (n=32) or virus (n=28) infections. All patients survived for more than 3 months after admission. In view of the fact that other clinical disorders or treatments may also affect the expression of certain genes, this study excluded patients with initiated therapy or the ones complicated with other clinical disorders. Healthy controls were subjected to systemic physiological tests and all physiological functions were within normal range. All participants signed informed consent.
Plasma and treatment
Before therapy, all patients and healthy controls were subjected to blood (3cm) extraction under fasting conditions. Plasma samples were prepared by centrifuging the blood samples for 15min at 1200g. All patients were treated with antibiotics in combination with oxygen and intravenous fluids. At 3 months after treatment, fasting blood was also extract from each patient to prepare plasma samples. RNAs were extracted from plasma samples immediately after plasma preparation.
Human Bronchial Epithelial Cells (HBEpCs)
With lung injury in sepsis as a focus, HBEpCs (Sigma-Aldrich) were used as the cell model. Cell culture medium was Bronchial Epithelial Cell Medium (Cat. #3211; PromoCell). A 95% humidity and 5% CO2 incubator (37°C) was used to cultivate cells. At passage 3 to 5, cells were harvested to perform following experiments. To explore the effects of LPS on gene expression, HBEpCs were cultivated in medium supplemented with LPS at doses of 0, 1, 2, 5, and 10 µg/ml for 48h before use.
Cell transfections
MALAT1 or CRNDE expression backbone vector was constructed with pcDNA3.1 vector (Invitrogen). MALAT1 or CRNDE expression vector (1μg) or empty vector (1μg, negative control (NC) group) was transfected into 108 HBEpCs using lipofectamine 2000 (Invitrogen). After transfections, cells were cultivated for further 48h before use. To perform control (C) experiment, cells without transfections were cultivated until the end of cell culture.
RNA preparations
Isolation of total RNAs from both plasma samples and HBEpCs was performed using Ribozol reagent (Invitrogen). RNA integrity was tested using 5% Urine PAGE gel. Genomic DNA removal was performed with gDNA eraser for 2h at 37°C. OD values at 260 and 280 were measured and the ratio of 260 to 280 was calculated.
RT-qPCRs
Total RNA samples with a 260/280 ratio close to 2.0 (pure RNA) were used as template to synthesize cDNAs through reverse transcriptions, which were performed using SSRT IV system (Invitrogen). With cDNA samples as template, qPCRs were performed using SensiFAST™ Real-Time PCR Kit (Bioline) with 18S rRNA as internal control to normalize the expression levels of MALAT1 and CRNDE. Three technical replicates were included in each experiment and gene expression levels were normalized using the method of 2-ΔΔCT.
Cell apoptosis assay
To induce cell apoptosis, HBEpCs with transfections were cultivated in medium supplemented with 10 µg/ml LPS for further 48h. Cell culture was performed in a 6-well cell culture plate with 8000 cells in 2 ml medium per well. Three wells were set for each experiment. After that, ice-cold PBS was used to wash cells and PI and FITC-annexin V (Sigma-Aldrich) were used to stain cells in dark for 20 min. After that, flow cytometry was used to separate apoptotic cells.
Statistical analysis
Mean±SD was used to express all data from 3 independent values. Gene expression levels in plasma samples were expressed as averaged values. Differences between two groups were analyzed by unpaired t test. Paired t test was used to compare two time points of the same group. Differences among multiple groups were analyzed by ANOVA Tukey’s test. Correlation analyses were performed using linear regression. P<0.05 was set to be statistically significant.