Cell Culture and source of miRNA
Human HCC HepG2 cells used was purchased from the Cell Bank of the Chinese Academy of Sciences by Kilton Biotechnology Co., Ltd. (Shanghai, China). HepG2 cells were cultured in a humidified incubator with 37℃ atmospheres supplemented with 5% CO2, using Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. miR-183-5p mimics, mimics negative control (NC), miR-183-5p inhibitor, inhibitor NC were purchased from Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). The sequence was listed in Table I.
Reagents and antibodies
Lip-2000 were obtained from Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China); ELISA reagent kit: Feiya Biological Technology Co., Ltd (Jiangsu, China); CCK-8 reagent kit: Yisheng Biotechnology Co., Ltd (Shanghai, China). Antibodies used for Western blot were as follows: FoxO1, NLRP3, caspase-1, GSDMD, IL-1β, IL-18 and GAPDH (GeneTex, USA; Servicebio, Wuhan, China). NLRP3, caspase-1, IL-1β, IL-18 and GAPDH were used in a dilution of 1:1,000. FoxO1, GSDMD were used in a dilution of 1:500.
Cell transfection
HepG2 cells were plated in 6-well plate with 1*106/ml. miR-183-5p mimics, mimics NC or miR-183-5p inhibitor, or inhibitor NC were transfected into cells, according to the manufacturer’s protocol. Firstly, configuration solution A (25 µL liposome lip-2000) and solution B (10 µL miR-183-5p mimics, mimics NC or miR-183-5p inhibitor, inhibitor NC) were added in 100 µL serum-free basal medium, then were incubate at room temperature for 4 h. The transfection medium was replaced with fresh DMEM medium containing 10% fetal bovine serum for further culture.
Cell counting kit 8 (CCK-8)
CCK-8 assay was performed according to the manufacturer’s instructions. Briefly, HepG2 cells were plated in a 96-well plate with 1*104/mL, miR-183-5p mimics, mimics NC or miR-183-5p inhibitor, or inhibitor NC were transfected into cells, then 10 µL of CCK-8 solution was added to each well of a 96-well plate and incubated at 37°C for 4 h. The optical density was measured at an absorption wavelength of 450 nm. Results were normalized to control levels.
IL-1β, IL-18 (ELISA)
Culture medium was collected according to manufacturer's protocols, IL-1β or IL-18 in the supernatant was measured, using commercial ELSIA Assay Kit. In brief, IL-1β, IL-18 substrate mix and assay buffer were added into cell culture supernatants in a 48 well plate. After the incubation at 37℃ for 15min without light, the absorbance was recorded at 450 nm on microplate reader.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from HCC cells, using TrizoL reagent according to the manufacturer’s instruction. A total of 2 µg of RNA was used for cDNA synthesis with a Reverse Transcription Kit in 20 µL reaction volume. Equal amounts of cDNA were subjected to PCR with the SYBR Green PCR Master Mix Kit. The condition including an initial denaturation at 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a terminal extension at 72°C for 5 min. Each sample was examined in triplicate and β-actin was used as the internal control. GraphPad Prism v8 was used to create the scatter plots. All primers were obtained from TaKaRa. The primers we used for qRT-PCR is listed in Table II.
Western blot analysis
Equal amount of proteins were extracted from cells and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the manufacturer’s instruction, and then transferred to a polyvinylidene difluoride membrane. After blocking the membrane with 5% non-fat milk, membranes were incubated overnight with the specific primary antibodies at 4°C, and then incubated for 1h with the corresponding secondary antibody. The band intensities were analyzed and protein expression was quantified using Alpha EaseFC.
Statistical analysis
Statistically significant differences were assessed by Student’s t-test or one-way analysis of variance tests followed by Bonferroni’s method or Fisher's Least Significant Difference for multiple comparisons when necessary. The data were expressed as the mean ± SD. Coefficients of correlation (r) were determined by the Pearson correlation method. The criterion for statistical significance was set at P < 0.05.