2.1. Chemicals, reagents and animals
Polyphyllin B (purity>98%) was purchased from Herbest (Baoji, Shaanxi, China, LOT.NO.HR22111W3), the molecular formula of which is C51H82O20. PPB was dissolved with dimethyl sulfoxide (DMSO, BioFroxx, Guangzhou, China) and RPMI-1640 medium (Servicebio, Wuhan, China) (Concentration of DMSO in the medium <0.1%).
N-cadherin (ZEN-BIOSCIENCE, LOT.KK0510), E-cadherin (ZEN-BIOSCIENCE, KK0426), Vimentin (ZEN-BIOSCIENCE, 0506), Snail (Bioss, AF08174021), Twist (Bioss, AH09191908), MMP-9 (Cell Signaling, 13667S), Zeb1 (Abcam, GR3215620), MMP-2 (Bioss, 140529), GAPDH (Zsbio, 210040421), ki-67 (MXB Biotechnologies, 2203170672C2). The goat anti-rabbit lgG (Zsbio, 140193) and goat anti-mouse lgG (Zsbio, 202700514). Hematoxylin (Ebiogo, 09232110), Eosin (Ebiogo, 09122109), Fontana-Masson Stain Kit (Abcam, GR3335375-1).
All C57BL/6 mice (male,5-6 weeks old) were acquired from Hangzhou Ziyuan Experimental Animal Technology Co,.Ltd. (Hangzhou, China, SCXK2019-0004). All procedures for animal experiments were performed following the “Principles of Laboratory Animal Care” and guidelines of the laboratory animal care committee of Anhui University of Chinese Medicine (Animal Ethics Number: AHUCM-mouse-2021062).
2.2 Cell culture
The melanoma cell lines B16F10 was kindly provided by the Stem Cell Bank, Chinese Academy of Sciences (Beijing, China), and cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS, Gibco, CA, U.S.A.), 100 units/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2.
2.3 Transcriptomic sequencing of LncRNA
6 C57BL/6 mice (male, 18-20g, 5-6 weeks old) were housed under specific pathogen-free conditions at a temperature of 24 ± 1˚C and humidity of 55 ± 5% in a laminar airflow room with a 12 hours light and 12 hours dark circle. After acclimatization for 1 week,6 mice were subcutaneously inoculated at the right axilla with 1 × 106 B16F10 cells per 100 μL PBS to produce subcutaneous graft tumor model. After 1 day of tumor inoculation, B16F10 cells injected-mice were randomly divided into two groups: 3 mice + 100 μL distilled water/2d (the control group); 3 mice + 100 μL PPB 1mg/kg/2d (the PPB group). After 7 days of tumor inoculation, one intraperitoneal injection was given every other morning for 14 days. 14 days after starting the administration, mice were anesthetized by intraperitoneal injection of 100 μL of 1% sodium pentobarbital solution, cervically dislocated and executed, and the tumors were immediately surgically removed. Three replicates of each group were set up, trizol was added, total RNA was extracted, and the samples were sent to Tsingke Biotechnology Co., Ltd (Beijing, China). After passing the quality control, the whole transcriptome library was constructed for the tested samples, and PE150 sequencing was performed through Illumina platform.
2.4 Small interfering rna (siRNAs) and transfection
Incubate 2 × 105 cells/well in 6-well plates, after reaching 60-70% confluency, over-NC (NC) cells and over-NEAT1 (OE) cells were obtained by transfecting B16F10 cells using lentivirus (Hanbio Biotechnology, Shanghai, China) at 37°C, 5% CO2 according to the manufacturer's instructions. The B16F10 cells used in the follow-up experiments were B16F10 NC cells and OE cells. The sequence of NEAT1 over-RNA is shown in Supplementary Material. The mapping of the overexpression vector LV-m-NEAT1 is shown in Supplementary Figure 1. Micrographs of NC cells and OE cells under fluorescence microscopy and results of verification of overexpression levels using QRT-PCR assays are shown in Supplementary Figure 2.
2.5 Cell viability assay
Cell viability was assessed using MTT. NC cells and OE cells were inoculated into 96-well plates at a density of 6 × 103 cells/well and cultured overnight. Cells were treated with different concentrations of PPB (DMSO<0.1%) in each well for 24h and 48h, and then incubated with 20 μL of MTT (50 μg/mL final concentration ) for 4h in each well. Following solubilization with DMSO, the absorbance values were then measured at 570 nm. Cell viability = (absorbance value of PPB-treated group/absorbance value of control group) × 100%, and the IC50 for 24h and 48h was calculated by entering the cell viability of each group into Graphpad 8.0.2.
2.6 Wound healing assay
The effect of PPB on cell migration was assessed using a wound healing assay. NC and OE cells were inoculated at a density of 5 × 105 cells/well and cultured for 24h. After scraping the cell monolayer with a sterile micropipette tip, the wells were washed with PBS and treated with medium or PPB (1.39 μM) (DMSO<0.1%) for 24h. After drug treatment, pictures were taken under the microscope (200×) at 0h and 24h, and then the healing area was measured using Image J 6.0 software. The wound healing rate was calculated as: Healing rate = (wound area at 0h - wound area at 24h)/wound area at 0h×100%.
2.7 Transwell migration and invasion assay
The migration and invasion of NC and OE cells was assessed in transwell chambers containing 24 mm diameter polycarbonate membranes. The NC and OE cells (2 × 105 cells/well) were seeded in the upper chamber without matrigel coating for the migration assay and in the upper chamber precoated with matrigel for the invasion assay. After cells were treated with medium or PPB (1.39 μM) (DMSO<0.1%) for 24h, cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet, and then the wells were washed with PBS. Counted and photographed with a microscope.
2.8 Establishment of mouse subcutaneous transplantation tumor model and mouse melanoma lung metastasis model
24 C57BL/6 mice (male, 18-20g, 5-6 weeks old) were housed under the same conditions as in 2.3. After 1 week of domestication, 12 mice were each injected with 100 μLPBS containing 1×106 B16F10 NC cells mixed in the right axilla, and another 12 mice were each injected with 100 μLPBS containing 1×106 B16F10 OE cells mixed in the right axilla to establish a mouse melanoma subcutaneous transplantation tumor model, and D1 was recorded on the day of inoculation. After 1 d of tumor cell inoculation, the mice were randomly divided into 4 groups of 6 mice each: 6 mice inoculated with NC cells+100 μL saline/2d (NC control group); 6 mice inoculated with NC cells+100 μL PPB 1mg/kg/2d (NC administration group), 6 mice inoculated with OE cells+100 μL saline/2d (OE control group), and 6 mice inoculated with OE cells+100 μL PPB 1mg/kg/2d (OE administration group). All mice were weighed every other day, and PPB solution prepared with saline was injected intraperitoneally once every other morning for 14 days after 7 days of tumor inoculation.14 d after initiation of drug administration, all mice were sacrificed by cervical dislocation. The tumor samples were immediately surgically removed, then weighed and photographed, and stored in -80℃ refrigerator and 4% paraformaldehyde.
A lung metastasis model of melanoma was established using 100 μL PBS containing 1×106 B16F10 NC cells or OE cells mixed in the tail vein of 12 C57BL/6 mice. 3 mice/group,the grouping, administration and anesthesia were the same as described above. Mice are executed and bone, liver and lung tissue are immediately surgically removed and the lungs are photographed with a camera.
2.9 Flow cytometry analysis of B16F10 cells metastasis in vivo
Detection of Lungs and Liver metastases by B16F10 cells: fresh lung and liver tissues from 2.8 were ground, filtered, lysed of red blood cells, and then washed and centrifuged to obtain cell homogenates for flow detection of GFP fluorescence intensity. Detection of B16F10 cells on bone metastasis: remove the femur and tibia of one of the legs of each group of mice in 2.8, then rinse the bone marrow cavity with a syringe (replaced with a 1 mL needle) aspirated with 2 mL of 1640 medium, flush out the bone marrow, centrifuge the lysed red blood cells and wash again to obtain whole bone marrow to detect GFP fluorescence intensity.
2.10 Western blot analysis
Cells were collected after treatment with medium or PPB (1.39μM) (DMSO<0.1%) for 24h; the tumor samples were removed from the -80°C refrigerator, ground under an ice bath to obtain a homogenate. The protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 2 hours and then reacted with the primary antibody (1:1000) overnight at 4°C. After washing with TBST, the membranes were incubated with the goat anti-rabbit lgG or goat anti-mouse lgG (1:10000) for 2 h at 4℃.
2.11 HE, melanin and immunohistochemistry staining
HE staining: tumor samples were dehydrated and embedded, followed by deparaffinization and hematoxylin staining for 15 min. Next we treated the tissue with hydrochloric acid and alcohol for 10 s and placed it in an ammonia solution. Finally, the tissues were stained with eosin for 5 min.
Melanin staining using fontana-masson stain kit. Tumor samples were incubated in warm Amino Silver Solution, Gold Chloride Solution (0.2%), Sodium Thiosulfate Solution (5%), and Nuclear Fast Red Solution, respectively, for melanin staining according to the manufacturer's instructions.
Immunohistochemistry staining: the tumor samples were removed from 4% paraformaldehyde, embedded in paraffin, cut into thin slices and dewaxed. The slices were soaked in the order of 100%-95%-80% concentration of ethanol for 5 min each time and washed with distilled water. Slices were incubated with 3% H2O2 for 20 min at room temperature to remove endogenous peroxidase activity, and then the slices were washed with PBS. The slices were placed into primary antibodies and incubated overnight at 4°C. The primary antibodies (1:300) comprised MMP2, MMP9, Vimentin, E-cadherin, N-cadherin and ki-67. Subsequently, the slices were washed with PBS and incubated with secondary antibodies (1:3000) for 30 min at 37°C. Finally, the slices were stained with aminobenzidine (DAB), rinsed with distilled water and stained with hematoxylin for 1 min.
The slices were photographed under a light microscope in three random high-power fields. The Image Pro-Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze the images. The black color developed in the melanin staining and brown color developed in the immunohistochemistry test was set as the unified standard for judging the positive reaction of all photographs, and each photograph was analyzed to obtain the integral optical density (IOD) and the pixel area of the tissues (AREA) for each photograph. Then, the average optical density values (AOD) were calculated using the formula AOD = IOD/AREA.
2.12 Statistical analysis
Quantitative study results were plotted using GraphPad Prism 8.0.2 software. All experiments were repeated at least three times. Data were compared using one-way analysis of variance (ANOVA) and Dunnett's test, and P<0.05 was considered a statistically significant difference.