Cell culture
Our study used ATDC5 cell line (BeNa Culture Collection: BNCC350793), which are derived from the mouse teratocarcinoma strain AT805.
After the primary cell attachment have reached 85%-90%, the cell passage begins. We add digestion solution containing 0.25% trypsin: 0.01% EDTA (1:1) for 1 min. Wait until the cells have shrunk and become rounded and the gap has increased before adding complete medium to stop the digestion. After the cells have shrunk, become rounded, and gaps have been increased, complete medium is added to stop the digestion. We collected the cell suspension and centrifuged for 4 minutes, leaving the bottom cell cluster. Then add fresh complete medium and mix well by pipetting. The cells were inoculated into multiple petri dishes at 2×105/ mL, and cultured in a cell incubator with a constant temperature and saturated humidity (5% CO2 at 37°C).
Cell transfection
Seed the cells in a 6-well plate. When the cells are about 80% confluent, Lipofectamine™ 2000 transfection reagent (thermofisher, 11668019) was used, and the experiment was carried out according to the kit instructions. After 48 hours of transfection, the cells were collected and used for further analysis.
Plasmid construction
The recovered and purified target fragment HMGB1-3UTR (XhoI/NotI) was linked with the pYr-MirTarget (XhoI/NotI) vector. The product was named pYr-MirTarget-HMGB1-3UTR.
Detection of gene expression level by RT-PCR method
Total RNA was extracted from the cells using TRIzol (Sigma, T9424-100ML). In our study, the expression level detection mainly included miRNA, HMGB1, Bcl-2, Bax, P62 and Beclin1.
The real-time fluorescent quantitative PCR kits used in this study include Takara's TB Green^TM Premix ExTaq^TM II (Tli RNaseH Plus) and PrimeScriptTM RT Master Mix (Perfect Real Time), and TIANGEN's miRcute enhanced miRNA cDNA first-strand synthesis kit and miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green). The specific experimental steps are carried out according to the instructions. All primers required for RT-PCR in this study were summarized in Table 1.
Protein detection (Western blot)
The main proteins tested include HMGB1, the apoptosis-related protein Bcl-2/Bax, autophagy-related protein P62 and Beclin1. All antibodies used in this study are from Proteintech
The cells were washed with pre-cooled PBS (phosphate buffer), then 50 μL cell lysate (RIPA) was add to each well. We transferred the cell-containing lysate to a 1.5 mL EP tube, sonicated it on ice for 1 h and then centrifuged at low temperature for 30 min. Finally, the supernatant (cell protein lysate) was transferred to a new EP tube.
Specific steps: polyacrylamide gel for electrophoresis, transfer membrane (wet transfer method: transfer protein on the gel to the nitrocellulose membrane), wash membrane (0.05%TBST), milk blocking (5% skimmed milk powder, room temperature 1h), incubate primary antibody, wash membrane again, incubate secondary antibody (HRP-labeled antibody), and color development (ClarityTM Westren ECL Substrate, Bio-RAD).
Dual luciferase detection report related to the regulation of HMGB1 and miRNA
We used bioinformatics related tools (TargetScan) to predict the target genes of miR-142-3p. In this study, primers were designed based on the predicted binding sites of miR-142-3p and target genes or mutations containing binding sites (target gene 3’UTR, Table 1). We amplified the 3’UTR sequence and constructed a 3’UTR luciferase reporter gene vector. Plasmid and miRNA were co-transfected into 293T cells, and the fluorescence intensity was measured by Promega GloMax 20/20 Luminometer.
Cell proliferation assay
CCK-8: Cells were inoculated to 96-well cell culture plates in the form of 1×105/mL. When the cells adhered to the wall and grew to about 90%, the cells were transfected according to different test groups. Change to complete medium after 6 hours. Discard the supernatant after 24 hours, then add the CCK-8 solution (dilute the medium and the CCK-8 stock solution at a ratio of 1:9), and react for 4 hours. Finally, the OD value was detected at 450nm in the microplate reader.
Apoptosis detection
FITC-Anexin-V/PI double staining method: cells are s inoculated to 6-well cell culture plate. When the cells adhere to the wall and grow to about 105-106, the cells are transfected according to different groups. Change to complete medium after 6 hours, digest with trypsin after 24 hours, and wash with PBS. Then discard the supernatant and add 500μL Binding Buffer to resuspend the cells.
Finally, according to the kit (Keygen BioTBCH: KGA108-2) instructions, add 5μL Annexin V-FITC and 5μL Propidium lodide staining solution respectively, and test on the flow cytometer.
Cell migration and cell cycle detection
The cell scratch method was used to detect the cell migration rate. The cell cycle is detected using a cell cycle detection kit (Keygen BioTBCH). The experimental operation was performed according to the kit instructions. Finally, use flow cytometry for detection and analysis. The data was sorted and analyzed using the cell cycle fitting software ModiFit.
Statistical analysis
All experimental data in our study were obtained through three repeated independent experiments, and the results are expressed as ‘mean ± standard deviation’. According to the experimental conditions, t-test or ANOVA was selected to evaluate whether it is statistically significant (p <0.05 indicates significant and statistically significant).