Sepsis mouse model
The sepsis mouse model was established in BALB/c mice by the treatment of lipopolysaccharide (LPS, sigma, USA). The BALB/c mice (male, four-week-old) were purchased from the Academy of Military Medical Sciences (Beijing, China). Briefly, the BALB/c mice (male, four-week-old) (n=5) were saved in a humidity- and temperature-regulated place under the 12-hours usual dark/light circle with water and food. The mice were intraperitoneally injected with LPS (20 mg/kg) or the equal volume of saline. The mice were injected with the lentivirus carrying the CASC7 shRNA or corresponding control through tail vein. The lentivirus carrying the CASC7 shRNA corresponding control were obtained (GenePharma, China). The mice were euthanatized by cervical dislocation (CD) and harvest liver tissue and plasma samples for further analysis. The liver injury was analyzed by the Hematoxylin and Eosin (HE) staining in the mice. The levels of TNF-α and IL-1β were measured by the ELISA assays in the mice. Animal care and method procedure were authorized by the Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences Animal Ethics Committee.
Cell culture and treatment
The human liver LO2 cells were purchased in American Type Tissue Culture Collection. The cells were cultured in the medium of DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 0.1 mg/mL streptomycin (Solarbio, China) and 100 units/mL penicillin (Solarbio, China) at a condition of 37 °C with 5% CO2. The lentivirus carrying the CASC7 shRNA, miR-217 mimic, CASC7 overexpression vector, TLR4 overexpression vector and corresponding control were obtained (GenePharma, China). The transfection in the cells was performed by Liposome 3000 (Invitrogen, USA) according to the manufacturer's instructions. The levels of TNF-α and IL-1β were measured by the ELISA assays in the cells.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
The apoptosis was analyzed by using the TUNEL detection kit (Roche, Germany) in the liver tissues of the mice according to the product’s guidance. After the staining of TUNEL, the ventricular samples were dyed by DAPI (Sigma, USA) to stain nuclear. Fluorescence was observed using a confocal microscope (Olympus Fluoview1000, Tokyo, Japan).
CCK-8 assays
The cell viability was analyzed by the CCK‐8 assays. About 5×103 cells were put into 96 wells and cultured for 12 hours. Then the cells were used for the transfection or treatment. After 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours, the cells were added with a CCK-8 solution (KeyGEN Biotech, China) and culture for another 2 hours at 37°C. The ELISA browser was applied to analyze the absorbance at 450nm (Bio-Tek EL 800, USA).
Analysis of cell apoptosis
Approximately 2×105 cells were plated on 6-well dishes. Cell apoptosis was analyzed by using the Annexin V-FITC Apoptosis Detection Kit (CST, USA) according to the manufacture’s instruction. Briefly, the cells were collected and washed by binding buffer (BD Biosciences, USA) and were dyed at 25 ℃, followed by the flow cytometry analysis.
Quantitative reverse transcription-PCR (qRT-PCR)
The total RNAs were extracted by TRIZOL (Invitrogen, USA) from the tissues of the mice and cells. The first-strand cDNA was synthesized using Stand cDNA Synthesis Kit (Thermo, USA) as the manufacturer's instruction. The qRT-PCR was carried out by applying SYBR Real-time PCR I kit (Takara, Japan). The standard control for mRNA/lncRNA and miRNA was GAPDH and U6, respectively. Quantitative determination of the RNA levels was conducted by SYBR GreenPremix Ex TaqTM II Kit (TaKaRa, Japan). The primer sequences are as follows:
CASC7 forward: 5′-ATCAACGTCAAGCTGGGAGG-3′
CASC7 reverse: 5′-CTTGTCCCCCGCTCGTTC-3′
TLR4 forward: 5′-TGGATACGTTTCCTTATAAG-3′
TLR4 reverse: 5′-GAAATGGAGGCACCCCTTC-3′
miR-217 forward:
5′-CATGCTCGAGCTTATCAAGGATAAAATACCATG-3′
miR-217 reverse:
5′-GTTACGGCCGCTTGAGATCTACTCTAATTTCTTTTTTAAC-3′
GAPDH forward: 5′-AAGAAGGTGGTGAAGCAGGC-3′.
GAPDH reverse: 5′-TCCACCACCCAGTTGCTGTA-3′
U6 forward: 5′-GCTTCGGCAGCACATATACTAA-3′
U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′
Western blot analysis
Total proteins were extracted from the cells or tumor tissues of the mice with RIPA buffer (CST, USA). The concentrations of protein were analyzed by using the BCA Protein Quantification Kit (Abbkine, USA). Same concentration of protein was divided by SDS-PAGE (12% polyacrylamide gels), transferred to PVDF membranes (Millipore, USA) in the subsequent step. The membranes were hindered with 5% milk and hatched overnight at 4°C with the primary antibodies for PARP TLR4 (1:1000) (Abcam, USA), cleaved PARP (1:1000) (Abcam, USA), caspase3 (1:1000) (Abcam, USA), cleaved caspase3 (1:1000) (Abcam, USA) and GAPDH (1:1000) (Abcam, USA), in which GAPDH served as the control. Then, the corresponding second antibodies (1:1000) (Abcam, USA) were used for hatching the membranes 1 hour at room temperature, followed by the visualization by using an Odyssey CLx Infrared Imaging System. The results of Western blot analysis were quantified by ImageJ software.
Luciferase reporter gene assay
The luciferase reporter gene assays were conducted by applying the Dual-luciferase Reporter Assay System (Promega, USA). Briefly, the cells were treated with the miR-21 mimic or control mimic, the vector containing CASC7, CASC7 mutant, TLR4, and TLR4 mutant fragment were transfected in the cells by using Lipofectamine 3000 (Invitrogen, USA), followed by the analysis of luciferase activities, in which Renilla was applied as a normalized control.
RNA pull-down
Biotin-marked RNAs were transcribed by using biotin-UTP of MEGAscript T7 Kit (Thermo, USA) in vitro and purified by MEGAclear Kit (Thermo, USA) according to manufacturer’s guidance, and then incubated with entire cell lysates. Biotin-labeled transcripts and interacted RNAs were isolated with streptavidin beads and then subjected to qPCR analysis
Statistical analysis
Data were presented as mean ± SD, and the statistical analysis was performed by GraphPad Prism 7 software. The unpaired Student’s t-test was applied for comparing two groups, and the one-way ANOVA was applied for comparing among multiple groups. P < 0.05 were considered as statistically significant.