2.1. Cell lines and reagents
The TC cell lines, B-CPAP and C643, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were routinely cultured in RMPI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, USA) in an atmosphere of 5% CO 2 at 37℃.
An electuary ointment of Huaier extract was donated by Qidong Gaitianli Pharmaceutical Co., Ltd. (Jiangsu, China). Huaier extract contained four monosaccharides and 18 amino acids; its main active ingredients were polysaccharides, named TP-1 (fucose, arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 3.2:2.0:33.5:5.5:1.2:50.4), TP-2 (arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 7.4:4.5:2.7:9.4:1.9), TP-3 (fucose, arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 0.1:1.0:5.4:4.4:2.1: 7.0), and TP-4 (arabinose, galactose, glucose, xylopyranose, mannose, at molar ratios of 8.9:1.6:3.4:7.4:1.3).
2.2. Preparation of Huaier aqueous extract
A mass of 2 g Huaier ointment was dissolved in 20 mL complete medium, passed through a 22-µm filter, and stored at -20℃ for future use.
2.3. Determination of cell viability
The viability of B-CPAP and C643 cells in the presence of increasing concentrations of Huaier was measured using a cell counting kit-8 (CCK-8) kit (DOJINDO, Japan). An experimental group, control group, and blank group consisted of five replicate wells each. B-CPAP and C643 cells were seeded on 96-well plates at a density of 2 × 103 cells/well. Following synchronization, 200 µL medium containing 1, 2, 4, 8, 12, or 16 mg/mL Huaier was added, and the cells were cultured for 24 h, 48 h, or 72 h. Subsequently, 100 µL medium containing 10% CCK-8 was added to the cells and incubated for 3 h, following which the absorbance was measured at 450 nm using a microplate reader (Bio Tek, USA). The experiment was repeated three times.
2.4. Evaluation of apoptosis and the cell cycle
Cell apoptosis assay (Propidium iodide (PI)-Annexin V staining): The proportion of apoptotic cells was assessed using a BD Pharmingen™ PE Annexin V apoptosis detection kit (DOJINDO, Japan). Briefly, after treatment with 8 mg/mL Huaier extract for 48 h, B-CPAP and C643 cells were harvested, washed twice with PBS, and centrifuged at 1000 rpm for 3 min. Subsequently, 5 µL Annexin V-FITC and 5 µL PI solution were mixed with 1 × Annexin V binding solution (100 µL) containing cells at a density of 1 × 10 5/mL. Following incubation for 15 min in the dark at room temperature, another 400 µL 1 × Annexin V binding solution was added prior to analysis by FACScan flow cytometry (Becton-Dickinson, USA). The experiment was repeated three times.
Cell cycle assay: The cell cycle phases were evaluated using a cell cycle detection kit (KeyGen China). The experimental group was treated with 8 mg/mL Huaier extract, while the control group was incubated with complete medium. Cells were harvested after 48 h, rinsed twice with PBS, and centrifuged at 1000 rpm for 5 min. Subsequently, cells were fixed in cold 70% ethanol overnight, washed once with PBS the following day, resuspended in 100 µL RNaseA, and placed in water at 37℃ for 30 min. A volume of 400 µL PI staining solution was added and cells were analyzed by FACScan flow cytometry after incubation for 30 min at 4℃ in the dark. Data were analyzed using the ModFitLT V2.0 software (Becton-Dickinson). The experiment was repeated three times.
2.5. RNA extraction, library preparation, sequencing, and data processing
Following treatment with 8 mg/mL Huaier extract for 48 h, B-CPAP cells were harvested for high-throughput sequencing. Total RNA was isolated according to the manufacturer’s instructions. The quality and quantity of extracted RNA were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and an RNA 6000 Nano LabChip kit (Agilent Technologies, USA). The preparation of whole transcriptome libraries and deep sequencing were performed by Beijing Ori-Gene Science and Technology Corp., Ltd. (Beijing, China). The libraries were constructed using the Ribo-Zero Magnetic Gold kit (Illumina, USA) and the NEBNext® Ultra™ RNA Library Prep kit for Illumina (New England Biolabs) according to the manufacturer’s manual. Sequence reads were aligned to the human genome (GRCh38) using the TopHat 2.0 program, and the resulting alignment files were reconstructed by Cufflinks.
Following prediction of mRNAs and ncRNAs, histograms and box plots were used to represent the transcripts. A Pearson heat map was drawn to represent the difference between the two groups of samples. The differential genes were plotted using volcanoes and cluster analysis. GO and KEGG analyses were performed, and the co-expression network and ceRNA mechanistic diagram were constructed.
Total RNA was extracted, and cDNA was synthesized using a PrimeScriptTMRT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer's instructions. Subsequently, RT-qPCR was performed in three independent wells using SYBR Premix Ex Taq (TaKaRa, Japan), and relative RNA expression was calculated using the 2−ΔΔCt method. The primer sequences are shown in Supplementary Table 1.
2.6. shRNA lentiviral vector transduction and verification
The lentiviral vector was constructed by Shanghai Hanheng Technology Co., Ltd. A total of 30,000 cells/well were inoculated with lentiviral infection solution (MOI = 25), culturing to a fusion degree of 60 − 70%. After successful transduction, cells were screened to establish stable cell lines.
RT-qPCR was used to verify the expression of GADD45A. The specific primer sequences were as follows: human GADD45A forward: 5'-ACTGTCGGGGTGTACGAAG-3'; human GADD45A reverse: 5'-ATCAGGGTGAAGTGGATCTGC-3'; human GAPDH forward: 5'-GAAGGTGAAGGTCGGAGTC-3'; and human GAPDH reverse: 5'-GAAGATGGTGATGGGATTTC-3'.
Protein expression was verified by western blotting. Total cellular protein was extracted and quantitated using the Coomassie brilliant blue method. A 20-µL protein sample was separated by SDS-PAGE and transferred to PVDF membrane at 80 V for 1.5 h. The membrane was sequentially incubated in skimmed milk, specific primary antibody, and corresponding secondary antibody. Protein bands were visualized using C300ECL luminaire.
2.7. Evaluation of cell proliferation
B-CPAP cells were divided into control, B-CPAP-Scr (Scr), and B-CPAP-sh-GADD45A (shRNA) groups. Cell proliferation was evaluated using a CCK-8 assay, and PI single-staining and Annexin V-PE/7-AAD double-staining were used to assess the cell cycle and apoptosis, respectively, by flow cytometry.
For treatment with Huaier extract, B-CPAP cells were divided into B-CPAP-Scr (Scr), B-CPAP-Scr treated with 8 mg/mL Huaier extract (Scr-treated), and B-CPAP-sh-GADD45A treated with 8 mg/mL Huaier extract (shRNA-treated) groups. Cell cycle and apoptosis were evaluated as described above.
2.8. Dual-luciferase assay
GADD45A and ILF3-AS1 WT and mutant dual-luciferase plasmids were constructed. The dual-luciferase detection kit (Promega, USA) was used to measure gene activity according to the manufacturer’s instructions. Both the Firefly and Renilla values were measured and the Firefly/Renilla ratio was calculated. Each experiment was repeated three times.
2.9. Statistical analysis
Statistical analysis was performed using the SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA) software. The results are expressed as the mean ± standard deviation of at least three independent experiments in at least triplicate. One-way analysis of variance was used to assess significant differences between the groups; p < 0.05 was considered statistically significant.