Patients and clinical specimens
All human WT specimens were collected from the first and second hospital of Lanzhou University (Gansu, China). The samples collection has been approved by the Medical Ethics Committee of the first hospital of Lanzhou University (Gansu, China), and the informed consent of patients has been obtained. Bioinformatics database were used in this study: Gene-Cloud Biotechnology Information (GCBI):https://www.gcbi.com.cn/gclib/htmL/index, and Oncomine: https://www.oncomine.org/resource/login/htmL/index.
Cell line and cell culture
The human WT cell line G401 was obtained from the Cell Resource Center of Xiehe (Beijing, China). G401 cells were cultured in McCoy’s 5A medium (Sigma, USA) with 10% fetal bovine serum (HyClone, Logan, USA), 100 U/mL penicillin/streptomycin (Gibco) at 37°C in a humidified atmosphere with 5% CO2.
Immunohistochemistry
Immunohistochemistry (IHC) staining and scoring were based on previous methods [28]. Antibodies specific to ZMIZ1 (1:100; Abam, USA), Notch1 (1:200; ImmunoWay, USA ), Jagged1 (1:300; ImmunoWay, USA), Hes-1 (1:100; ImmunoWay, USA) and WT1 (1:500; ImmunoWay, USA) were applied according to the manufacturer’s protocols. In brief, paraffin sections (4μm) were deparaffinized in xylene and rehydrated through graded ethanol solutions. Next, the sections were processed by the conventional microwave heating in 0.01 M sodium citrate retrieval buffer (pH=6.0) for 5 minutes. Then, they were blocked by 10% normal goat serum for 20 minutes and incubated with primary antibodies and secondary antibodies. Finally, the sections were stained with 3, 3’-diaminobenzidine solution and counterstained with hematoxylin, and observed under the microscope (Leica Microsystems).
Wound healing assays
Cell migration was assessed by measuring the movement of cells in the scratched cell area. A scratch was created by a 1 mL pipette tube, and the photographs of wound closure were taken at 0, 24, 48, and 72 hours respectively. Migration was quantified by counting the total number of cells that migrated toward the original wound field.
Transwell assay
Invasion and migration assays were performed in 24-well Boyden chambers (Cornin, USA) in vitro. For performing invasion assay, matrigel-coated chambers (BD Biosciences, San José, CA, USA) containing 8 μm pores were used. Cells were seeded into the upper chambers (coated with matrigel) at a concentration of 2×105/ml in McCoy’s 5A medium containing 0.5% FBS. The lower chamber was filled with McCoy’s 5A medium containing 10% FBS as a chemoattractant. After incubation at 37℃ for 24 hours, non-invaded cells in the upper chamber were scraped off with a cotton swab. The successfully translocated cells were fixed with 10% formalin. Then, they were stained with 0.1% crystal violet for 30 minutes and counted under a light microscope. Cells from at least four randomly selected microscopic fields were counted.
In vivo migration assays
4 to 6-week-old male BALB/c nude mice (nu/nu) were purchased from the Animal Center of Beijing Province (Beijing, China). All animals are kept in SPF Animal Experimental Center of Lanzhou University in a 12-hour dark/light cycle, and sterile food and water were provided ad libitum. All experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Lanzhou University. We established a lung metastasis model in nude mice, 200 µL of cell suspension (1 ×107 cells/mL) were injected through the tail vein of each nude mouse in the experimental group (n=8/group). The amount of food consumed, water consumed, and the bodyweight of each nude mice were recorded every three days. Mice were sacrificed after eight weeks and the organs were dissected and collected simultaneously, including spleen, kidneys, liver, lungs, and heart.
Haematoxylin-eosin (H&E)
All primary organs were processed (fixed in 10% formalin for 24h, embedded in paraffin), and then 4 μm sections were prepared. Subsequently, consecutive tissue sections were stained with haematoxylin-eosin (H&E) to observe the metastatic nodules of organs under a microscope.
Reverse transcription (RT)-polymerase chain reaction (PCR) and real-time PCR
Total RNA was isolated from treated cells using TRIZOL reagent according to the manufacturer’s protocol and the complementary DNA (cDNA) was prepared from 2lg of total RNA with Superscript III reverse transcriptase (Invitrogen, CA, USA). Quantitative analysis of cDNA amplification was assessed by incorporation of SYBR Green nucleic acid stain (KAPA Biosystem) into double-stranded DNA. The primer sequences used in this study are shown in Table 1. PCR was performed on a total volume of 20 ll containing 20 ng of cDNA template, 0.4 ll each of forward and reverse primers, and SYBR Green PCR Master Mix. All cDNA samples were tested in triplicate using the Light Cycler 480. Data were presented as relative to control cells.
Western blot assay
Proteins were extracted using a lysis buffer and quantified using a bicinchoninic acid protein quantification kit (Solarbio, China). Cell lysates were separated using 8–12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Temecula, CA, USA). The membrane was then blocked in PBST solution containing 5% non-fat milk, and incubated at 4℃ overnight with the specific primary antibodies anti-ZMIZ1 (1:500; Abcam, USA), anti-Jagged1 (1:2000; ImmunoWay, USA), anti-GAPDH (1:5000; ImmunoWay, USA), and anti-Notch1 (1:500; ImmunoWay, USA), anti-HES-1 (1:1000; ImmunoWay, USA), anti-WT1 (1:1000; ImmunoWay, USA). The bands were detected using the Pierce ECL Western Blotting Substrate (Thermo Scientific, USA).
Statistical analysis
Statistical analysis was performed on Graphpad Prism7 software (GraphPad software, lnc., San Diego, CA). All experiments were carried out independently at least three times, and the data were presented as means ± SD. The differences among groups were calculated using one-way ANOVA or two-tailed student’s t-test.In all cases, P < 0.05 was considered statistically significant.