Cell line Human breast cancer MDA-MB-231 cells were purchased from the Shanghai Chinese Academy of Sciences Cell Bank.
2. Cell culture
Resuscitate the cells: quickly shake the cryotube containing 1mL of cell suspension in a 37℃ water bath to thaw, add 5mL of medium and mix well. Centrifuge at 1000RPM for 5 minutes, discard the supernatant, add 4-6mL complete medium and blow well. Then add all the cell suspension to the culture flask and culture overnight (or add the cell suspension to a 6cm dish), and culture overnight. Change the medium the next day and check the cell density. Cell passage: If the cell density reaches 80%-90%, it can be subcultured.
3. Cell processing and grouping
After drug treatment in MDA-MB-231 cell line (0uM, 40uM, 70uM, 100uM, 130uM, 160uM, respectively, treatment time points 12h, 24h, 48h, 96h), 3 replicate wells for each treatment concentration; protein extraction WB detection and RNA extraction for Q-PCR detection.
The cells treated with 160 uM were used as the observation object.
4. WB detection
Inoculate the MDA-MB-231 cells in the logarithmic growth phase in a culture flask. When the cells grow to 80%, discard the medium and add thymosin α1 (0uM, 40uM, 70uM, 100uM, 130uM, 160uM) with different concentrations, and treat The time is 12, 24, 48, 96h. Wash 3 times with PBS buffer, add cell lysate (RIPA: PMSF: Cocktail = 100: 1: 2) on ice for 30 min, 12 000 r·min − 1 centrifugation for 20 min, use BCA method to detect protein concentration, add Load the protein buffer solution, place it in boiling water for high-temperature denaturation for 8 min, take 100 µg of protein, separate it by 10% SDS-polyacrylamide gel electrophoresis, transfer it to PVDF membrane, and block with 5% BSA at room temperature for 40 min. The antibody was incubated overnight on ice, and the secondary antibody was incubated at room temperature for 60 min. Finally, the membrane was scanned with a dual-color infrared fluorescence scanning imaging system and images were collected. Each experiment was repeated 3 times.
5. Q-PCR detection
Inoculate the MDA-MB231 cells in the logarithmic growth phase in a culture flask. When the cells grow to 80%, discard the culture medium and add thymosin α1 (0uM, 40uM, 70uM, 100uM, 130uM, 160uM) containing different concentrations of thymosin α1 (0uM, 40uM, 70uM, 100uM, 130uM, 160uM) for treatment The time is 12, 24, 48, 96h. Wash 3 times with PBS buffer, extract the total RNA of MDA-MB-231 cells with the RNA extraction kit (Axygen), measure the content and purity of the total RNA with a nucleic acid detector, and use the Thermo reverse transcription kit to reverse 1 µg of the total RNA Recorded as cDNA. Carry out real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) with EGFR gene-specific primers, and operate according to the instructions. Reaction steps: 95 ℃ 10 min, 95 ℃ 15 s, 60 ℃ 1 min, a total of 40 cycles. Let 2-ΔΔCT represent the relative expression of the gene, and the internal reference gene is GAPDH.
6. Library preparation, sequencing, and data analysis
After the total RNA is processed to remove the DNA, the absorbance at 260nm/280nm (A260/A280) is measured to determine the quality and quantity of the purified RNA. 1.5% agarose gel electrophoresis further verified the integrity of RNA. Polyadenylated mRNAs are purified and concentrated with oligo (dT) bound magnetic beads (Invitrogen) before they are used in the preparation of directed RNA sequence libraries.The purified mRNAs are lysed in 95°C lysis buffer, the ends are repaired, and the 5'linker is ligated. Then RT primers containing 3'adaptor sequences and random hexamers were used for reverse transcription. Purify and amplify the cDNAs, purify the PCR products of 200ཞ500bps, quantify and store them at -80℃ for sequencing. Prepare following the manufacturer's instructions and use the Illumina HiSeq X Ten system for sequencing. The software we choose is edgeR for gene differential expression analysis. The analysis result uses fold change (FC) and false discovery rate (FDR) to determine whether a gene is differentially expressed. Significant difference expression evaluation criteria: FC ≥ 2 or ≤ 0.5, P-value < 0.01.
7. Wnt/β-catenin signaling pathway blockade
After the Wnt/β-catenin signaling pathway inhibitor, MSAB was used to treat the cells in the experimental group, Western blot was used to detect the expression of AXIN2, β-catenin, PD-L1, and C-myc.
8. Statistical analysis
The experimental result data is expressed as x̄ ± s, and the SPSS 26.0 software was used for statistical analysis. The comparison of means between groups was performed by t-test, and the comparison of means above two groups was performed by one-way analysis of variance.