Cell culture and transfection
Kidney podocytes cell line (Cat#GD-C8618339) were cultured in Roswell Park Memorial Institute Medium 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. Podocytes were cultured at 37˚C in a saturated humidified atmosphere of 5% CO2, with culture medium replacement every 2 d. The sequences of HBx were amplified and inserted into empty pcDNA3.1 vectors, yielding pcDNA-HBx (HBx). The same method was used to produce pcDNA-NLRP3 (NLRP3) and pcDNA-siNLRP3 (siNLRP3). Transient transfection with the indicated plasmids into podocytes was performed using Lipofectamine 2000.
Cell treatment with a miR-223 mimic or inhibitor
Podocytes were transfected with synthetic mature miR-223 (miR-223 mimic), antagomir antisense to mature miR-223 (miR-223 inhibitor), or a scrambled control transcript that served as a negative control. Subsequently, the expression levels of miR-223, NLRP3, ASC, caspase-1, IL-1β, and IL-18 were measured. In separate experiments, the podocytes were transfected and treated as described above, after which their apoptotic status was assayed.
Podocytes in six-well plates were fixed with 4% paraformaldehyde at room temperature for 30 min, incubated in phosphate-buffered saline (PBS) containing 0.4% Triton X-100 for 10 min, and blocked with 2% bovine serum albumin at 37°C for 60 min. The cells were then incubated with the primary antibodies anti-vimentin (1:100; Boster Biological Technology, Wuhan, China) and anti-keratin (1:100; Boster Biological Technology) at 4°C overnight. PBS was used as the negative control. Then, the cells were washed with PBS and incubated with anti-mouse IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 594 (1:1000; Life Technologies, Paisley, UK) for 1 h at room temperature. The glass cover slides were installed using the installation medium with 4ʹ,6-diamidino-2-phenylindole dye (Vector Labs, Peterborough, UK) and prepared for imaging under a fluorescence microscope under ⊆200 magnification (Olympus, Tokyo, Japan).
TargetScan and miRGator were used to predict the target gene of miR-223.
Dual luciferase reporter assay
Luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions.
Hoechst 33342 staining
The morphological changes of podocytes were observed by staining the nucleus with Hoechst 33342 (C0030; Solarbio, Beijing, China). The podocytes transfected with different vectors were stained with Hoechst 33342 and cultured in an incubator for 20-30 min, after which the medium was discarded, and the podocytes were washed with medium two to three times. The podocytes were then immediately observed and imaged under a fluorescence microscope.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA in cells was extracted using TRIzol reagent (Tiangen Biotech, Beijing, CHINA) and were reverse transcribed to cDNA using the SuperScript Reverse Transcription Kit (Tiangen Biotech). RT-PCR was performed using SYBR Green Master Mix (Tiangen Biotech) according to the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin served as the internal controls, and the 2−△△Ct method was used to calculate the relative gene expression. The primers sequences were listed as follows:
HBx forward primer: 5'-GCGAATTCATGGCTGCTAGGGTGTGCT-3'
HBx reverse primer: 5'-ATCTCGAGTTAGGCAGAGGTGAAAAAGTTGC-3',
NLRP3 forward primer:: 5'-CACCTGTTGTGCAATCTGAAG-3'
NLRP3 reverse primer: 5'-GCAAGATCCTGACAACATGC-3'
ASC forward primer: 5'-AGGCCTGCACTTTATAGACC-3'
ASC reverse primer: 5'-GCTGGTGTGAAACTGAAGAG-3'
Caspase-1forward primer: 5'-CCTTAATATGCAAGACTCTCAAGGA-3'
Caspase-1 reverse primer: 5'-TAAGCTGGGTTGTCCTGCACT-3'
IL-1βforward primer: 5'-TACCTGTCCTGCGTGTTGAA-3'
IL-1βreverse primer: 5'-TCTTTGGGTAATTTTTGGGATCT-3'
IL-18 forward primer: 5'-TGCATCAACTTTGTGGCAAT-3'
IL-18 reverse primer: 5'-ATAGAGGCCGATTTCCTTGG-3'
β-actin forward primer: 5'-AGCGAGCATCCCCCAAAGTT-3'
β-actin reverse primer: 5'-GGGCACGAAGGCTCATCATT-3'
GAPDH forward primer: 5'- GCTCGCTTCGGCAGCACA -3'
GAPDH reverse primer:5'- GAACGCTTCACGAATTTGCGTG-3'
Western blot (WB) analysis
Proteins were extracted from hematopoietic progenitor cells using radioimmunoprecipitation assay buffer, and their concentration was determined using the bicinchoninic acid method. After denaturation, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated overnight at 4˚C with the primary antibodies specific for: NLRP3 (1:1000; Abcam, Cambridge, UK), caspase-1, ASC, IL-1β, IL-18 (1:1000; ABclonal Biotechnology, Wuhan, China), and GAPDH (1:5000; Proteintech, Chicago, IL, USA). The membranes were then incubated with goat anti-rabbit or -mouse IgG (1:5000; ZSGB Biotech, Beijing, China) and quantified using ImageJ.
Cell pyroptosis was analyzed by flow cytometry using In Situ Cell Death Detection Kit (Immunochemistry, Bloomington, United States) according to the manufacturer's instructions. Podocytes were cultured in sixwell plates and divided into nine groups: control, empty plasmid, HBx, HBx + miR-mimic, HBx + miR-inhibitor, HBx + miR-mimic + NLRP3, HBx + miR-mimic + si-NLRP3, HBx + miR-inhibitor + NLRP3, and HBx + miR-inhibitor+ si-NLRP3 groups. After transfection, the podocytes were digested and collected in tubes. The supernatant was discarded after centrifugation followed by three washes and were double-stained with annexin V-fluorescein isothiocyanate and propidium iodide at room temperature for 15 min in the dark. The results were analyzed using flow cytometry, and pyroptosis rate was calculated as the percentage of early and late pyroptosis cells.
Measurement of caspase-1 activity
To determine the activity of caspase-1 in different groups, the cells were analyzed using a Caspase 1 Activity Assay Kit (Meilunbio, Dalian, China) according to the manufacturer's instructions.
Measurement data are expressed as the mean ± standard deviation, which were further analyzed by GraphPad Prisem 8.0. For normally distributed data, unpaired t-test was used to compare two groups. All experiments were performed at least three times. Statistical significance was set at a P-value < 0.05.