Chemicals and reagents
Huaier crude extract was obtained from Qidong Gaitianli Pharmaceutical Co., Ltd. (Qidong, China). Polysaccharides of Trametes robiniophila Murr (PS-T) were isolated and purified as previously reported by our team [41]. The purity of the PS-T was over 99%, which was evaluated using a phenol–sulfuric acid method with glucose as the standard [42]. Primary antibodies against MAP1LC3B (NB100-2220), Snail (NBP2-50300) were purchased from Novus Biologicals. E-cadherin (Ab40772), Cytokeratin (Ab7753), Vimentin (Ab8978) and ATG12 Antibody (ab155589) were from Abcam. ATG5 Antibody (PA5-23186) were from Thermo Fisher. N-cadherin (AF4039), Fibronectin (AF5335), Occluding (AF7504), P62 (AF5128), Beclin-1 (AF5128) and β-actin (AF7018) was purchased from Affinity. SNAI1 (SC-271977), and E-cadherin (SC-7870) were from Santa Cruz Biotechnology. Secondary antibodies against rabbit IgG (7074) were from Cell Signaling Technology. Secondary antibodies for immunocytochemistry (FITC and Cy3) were purchased from EarthOx. Other materials were obtained from commercially available chemicals.
Cell culture and CCK-8 assay
Human breast cancer MDA-MB-231 cells, mouse breast cancer 4 T-1 cells and human embryonic kidney HEK-293 T cells were purchased from FuHeng Cell Center, Shanghai, China. The cells were cultured according to their instructions. Cell viability was investigated by the CCK-8 assay. The experiment was conducted in accordance with the instructions.
Scratch assay, transwell migration and matrigel invasion assays
Confluent monolayers of cells were scratched with a 200 μL pipette tip. Cells were cultured in serum-free medium with or without PS-T (1 μg/mL or 5 μg/mL). The width of the scratch was measured using an Olympus inverted microscope at 0 h, 12 h, and 24 h. For the migration assay, transwell filters purchased from Corning (Corning Costar, USA) were used. Cells were incubated in serum-free medium with or without PS-T (5 μg/mL) for 12 h, then we suspended the cells (5 × 104) in 400 μL of serum-free medium and added to the upper chamber. Then, complete medium containing 10% fetal bovine serum was added to the lower chamber. After incubated at 37 °C for 24 h, the cells were stained with crystal violet and counted in three random fields. For the invasion assay, the transwell filter were covered with Matrigel (Corning, 354480).
In vivo metastasis assays
Six-week-old Balb/c female mice were purchased from the Model Animal Research Center of Nanjing University. All mice were housed for 3 days to acclimatize them to the conditions. 4 T-1 cells were injected into the lateral tail vein (1 × 106 cells per mouse). Mice were randomly divided into three groups (control, 25 μg/g PS-T, 100 μg/g PS-T) with five mice per group. The next day, the drug was administered by oral gavage every other day. The control group was treated with 100 μL of normal saline. Mice were sacrificed after 21 days. Lungs were harvested and fixed with Bouin’s solution. The lung metastatic nodules were counted by the naked eye. The scale includes four grades: grade I (nodule diameter < 0.5 mm), grade II (0.5 mm ≤ nodule diameter < 1 mm), grade III (1 mm ≤ nodule diameter < 2 mm), grade IV (nodules diameter ≥ 2 mm). Metastatic nodule score was calculated as follows: I × 1 + II × 2 + III × 3 + IV × 4. All of the research involving animals complied with protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (AMUWEC2019199).
Western blotting
Cells were washed with cold PBS and then harvested. The protein was extracted and the concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific), then stored at − 20 °C. Proteins were electrophoresed by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Signals were detected by an ECL detection system, and evaluated using ImageJ software.
mRFP-GFP-LC3 fluorescence for tracing autophagic flux
MDA-MB-231 cells were transfected with mRFP-GFP-LC3 plasmid according to the manufacturer's instructions and treated with 5 μg/mL PS-T or PBS for 24 h. Autophagy. The RFP and GFP puncta in cells was observed using laser confocal microscopy and counted to evaluate the level of autophagy.
Construction of ATG5 stable interference cell line
Three siRNAs targeting ATG5 mRNA at positions 393, 443, and 853 were designed. The interference efficiency of targeting small fragments was analyzed by qPCR. The results showed that siRNA targeting 853 was the most efficient, reaching 70% of interference. A small fragment of siRNA was inserted into the pLVX-ShRNA2-Puro plasmid to construct a lentiviral recombinant plasmid. The constructed plasmid was named pLVX-ShRNA2 -Puro-Homo-ATG5-853, and it was packaged and transferred into HEK293 cells. Transfection efficiency was observed at 24 and 72 h after transfection, and the lentivirus pLVX-ATG5 titer was measured as 8 × 107 TU/mL. MDA-MB-231 cells were infected, and infection efficiency was observed after 48 h. We then screened positive cells using the optimal concentration of 0.2 μg/mL puromycin. Monoclonal cells were obtained by limiting dilution (Additional file 2: Figure S2).
E-cadherin promoter activity assay
First, we looked up the information of E-cadherin on the chromosome in the Ensemble database (https://www.ensembl.org/index.html) and obtained the E-cadherin promoter sequence. pUC57-E-cadherin and pGL3-basic were double digested with SacI and HindIII, and then the recovered and purified target fragment E-cadherin (SacI/HindIII) was ligated to the vector pGL3-basic (SacI/HindIII), and the linked product was named pGL3 -basic-E-cadherin(Additional file 4: Figure S4). Then DH5a competent cells were transformed with the ligation product, coated with LB AMP + plates, and cultured in a 37 °C incubator overnight. A single colony was picked, inoculated in LB AMP + liquid medium, and cultured at 37 °C and 250 rpm overnight. The cultured bacteria liquid was used for colony PCR identification. Extraction was then performed using an endotoxin-free plasmid extraction kit and the extracts were stored at − 20 °C. MDA-MB-231 or MDA-MB-231-siATG5 cells were plated into 96-well plates at a volume of 3 × 104 cells/well in a volume of 100 µL and incubated overnight at 37 °C in an incubator. Subsequently, Lipo3000 was used for plasmid transfection. After 6 h, the solution was changed and 5 µg/mL PS-T was added for stimulation. After 24 h, the intracellular dual fluorescence was detected using the Promega Dual-luciferase assay kit (E1910) kit. The relative expression of firefly and renilla luciferase was calculated.
RNA isolation and quantitative RT-PCR
Total mRNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA), and followed by cDNA synthesis with Taq-Man Reverse an RNeasy kit (QIAGEN, Hilden, Germany). mRNA expression was detected using SYBR Premix Ex Taq II Kit (Takara, Kusatsu, Shiga Prefecture, Japan) in an Eppendorf Mastercycler realplex. The 2−∆∆Ct formula was used to calculate the relative abundance of RNA genes compared with GAPDH.
Immunofluorescence staining
Cells were cultured on coverslips for 24 h with or without 5 μg/mL PS-T. Cells were fixed with 4% (w/v) paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 20 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, followed FITC- or Cy3-conjugated secondary antibodies (1:100 in PBS) at room temperature for 60 min. The nuclear were counterstained using DAPI. For tissue immunofluorescence staining, rehydrated paraffin-embedded sections were microwaved in 10 mM sodium citrate buffer (pH 6) to unmask the antigen, and incubated with the primary antibody followed by Opal 520, 620, 570 secondary antibody for 60 min at room temperature. Images were captured by confocal microscopy (FV-1000; Olympus, Tokyo). The IPP6.0 software was used to perform optical density analysis on immunofluorescence photos. Three areas in 400 photos were selected per section to calculate mean optical density.
Lentivirus transfection
First, we queried the NCBI (https://www.ncbi.nlm.nih.gov/) and found the human Snail protein. The information is as follows: GenBank: AF125377.1, 264AA, coding sequence is 795 bp. This fragment was cloned into the pcDNA3.1 plasmid by BamHI/HindIII double restriction digestion. After transformation, clone selection, identification and sequencing, it was determined that the construction was successful. Extraction was then performed using an endotoxin-free plasmid extraction kit. Store at − 20 °C. Lipo3000 was used for pcDNA3.1-Snail recombinant plasmid transfection.
Bioinformatics
In this study, The Human Protein Atlas (https://www.proteinatlas.org) is used to compare protein expression of Snail in breast cancer tissues. The correlation between specific gene expression and survival curves in breast cancer was assessed by the GEPIA (http://gepia.cancer-pku.cn/), bc-GenExMiner 4.5 (http://bcgenex.ico.unicancer.fr), KM plotter (http://kmplot.com/analysis/). BRCA of TCGA was download from UCSC database (http://xenabrowser.net). We used the ratio of SNAI1/LC3B expression to stratify patients into high and low groups and looked for association with outcomes. In GEO gene-chip datasets, we used SNAI1(219480_at) as a numerator and LC3B(208786_s_at) as the denominator to calculate the SNAI1/LC3B ratio of each patient. In TCGA RNA-Seq datasets, we used the gene-level transcription estimates, as in log2(x + 1) transformed RSEM normalized count to calculate the SNAI1/LC3B ratio of each patient.
Immunohistochemistry (IHC) analysis
All patient samples were collected from the Breast Disease Center, First Affiliated Hospital of the Army Medical University (Chongqing, China). Specimens were fixed in 10% formalin and embedded in paraffin. After soaking in xylene to dewax and rehydrating using an ethanol gradient. The 4 μm slices were de-waxed, rehydrated and stained according to the SP kit instructions (ZSGB-BIO ORIGENE, SP-9000). IHC staining was scored according to the staining intensity score × the percentage of stained cells. The intensity of staining was scored according to 0 points: no staining, 1 point: weak positive, 2 points: moderate positive, 3 points: strong positive. The percentage of stained cells was counted 1 follows: 1–25%, 2 points: 26–50%, 3 points: 51–75% and 4 points: 76–100%.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8. All data were presented as means ± standard deviation and analyzed using variance (ANOVA) or Student’s t test. All experiments were performed at least three times. P < 0.05 was considered statistically significant.
