2.1 Microarray and transcriptome dataset analysis

The mRNA expression level of SAA1 was analyzed in 91 ESCC tissue samples and 11 normal esophageal tissue samples from The Cancer Genome Atlas (TCGA) database (http://www.cancer.gov/tcga). As a supplement, this study also analyzed the expression level of SAA1 mRNA in GSE161533 (28 paired normal tissues, paratumor tissues, and tumor tissues) and GSE44021 (73 paired normal tissues and tumor tissues) from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/).

2.2 Patients and tissue samples

Sixteen pairs of fresh tumor and normal tissues from 16 patients with ESCC were obtained from the Affiliated Hospital of North Sichuan Medical College. All tissues were rapidly frozen in liquid nitrogen after excision and then maintained at − 80 °C. This study was approved by the ethics committee of North Sichuan Medical College (NO. NSMC202161).

2.3 Cell culture and treatment

The human ESCC cell lines Eca109 and TE-1 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Science (Shanghai, China). A human normal esophageal epithelial cell line HEEC was obtained from ScienCell (CA, USA). All cells were cultured in RPMI 1640 medium (Gibco, United States) supplemented with 10% (v/v) fetal bovine serum (Every Green, China), 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, Austria). All cells were maintained in a standard humidified incubator with 5% CO2 at 37 °C.

2.4 Cell transfection and lentivirus infection

The siRNAs against SAA1 (SAA1-siRNA), S1PR1 (S1PR1-siRNA1, S1PR1-siRNA2) and the negative control (NC-siRNA) were synthesized by GenePharma (Shanghai, China). siRNA sequences for SAA1, S1PR1 and NC are shown in Table 1. The S1PR1 overexpression plasmid (OE-S1PR1) and control plasmid (OE-NC) were constructed by Genechem (Shanghai, China). Transient transfection of plasmids and siRNAs was performed with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. SAA1 overexpression (OE-SAA1) lentivirus and negative control (OE-NC) lentivirus were designed and produced by Genechem (Shanghai, China). TE-1 cells were infected with these lentiviruses and selected with puromycin (Solarbio, China).

2.5 RNA extraction and reverse transcription-quantitative PCR (RT‒qPCR) analysis

Total RNA was extracted using the SV Total RNA Isolation System (Promega, USA) following the standard procedure. The PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Japan) was used to obtain cDNA. RT‒qPCR analysis of the samples was performed using CFX Connect (Bio-Rad, USA) with a SYBR Premix ExTaq™ II (Tli RNaseH Plus) kit (Takara, Japan). HPRT was used as an endogenous control. The primers used are listed in Table 2. The relative expression of mRNA was computed using the 2−ΔΔCt method.

Table 2 RT qPCR primer sequences2.6 Cell counting kit-8 (CCK-8) assay

ESCC cells were seeded in 96-well plates. The next day, 40% confluent cells were transfected with siRNA. The cells were monitored every 24 h. CCK-8 (Beyotime, China) reagent was added to each well (at a 1:10 dilution). Cells were incubated for another 2 h at 37 °C. Then, the absorbance was measured at a wavelength of 450 nm by an iMark™ microplate reader (Bio-Rad, USA). The CCK-8 assay for the lentivirus-infected cells was the same as described above but without the transfection step after cells were seeded. In the recombinant human SAA1 (rhSAA1, PeproTech, USA) stimulation assay, TE-1 cells were stimulated with 0, 0.1, 1, and 10 µg/mL rhSAA1, and cell proliferation was monitored every 24 h.

2.7 Wound healing assay

Eca109 cells were plated in 12-well plates. After 24 h, the monolayer was scratched with a sterile pipette tip to form a “wound”. The debris was removed by washing twice with phosphate buffered saline (PBS), followed by cell transfection. Finally, images were taken at the same locations at 0, 24 and 48 h after scratching. The degree of cell migration at different times was quantified by the area between the two edges of the wound. The wound healing assay for the lentivirus-infected cells was the same as described above but without the transfection step after cells were seeded. In the rhSAA1 stimulation assay, TE-1 cells were treated with 0 or 10 µg/mL of rhSAA1, and cell migration was monitored for 24 h.

2.8 Colony formation assay

The cells were seeded into 6-well plates (800 cells/well) and cultured for 14 days in an incubator. Next, colonies were fixed with 4% paraformaldehyde for 60 min and stained with 0.5% crystal violet solution. The colonies in three random fields were counted, and the average was calculated.

2.9 Apoptosis assay

TE-1 cell apoptosis was determined using an Annexin V-APC/PI apoptosis assay kit (KeyGEN BioTECH, China) after transfection with SAA1 siRNA. Cells were digested with EDTA-free trypsin, subsequently washed with PBS, collected, and finally stained with Annexin V-APC and PI for 10 min in the dark. Cell apoptosis was analyzed using a flow cytometer (3L13C, ACEA Biosciences, USA).

2.10 Western blot analysis

Cells or tissues were lysed with ice-cold RIPA buffer containing a protease inhibitor mixture. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). Total protein was separated by SDS‒PAGE (8 or 15%). The designated proteins were transferred to pre-cut polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Next, the membranes were blocked in 5% bovine serum albumin (BSA)-TBST for 1.5 h at room temperature and incubated with specific primary antibodies at 4 °C overnight. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, China) for 60 min at room temperature and visualized with a ChemiDoc™ XRS + system (Bio-Rad, United States). ImageJ software was used to analyze the relative protein levels. Primary antibodies included those against SAA1 (1:1,000, R&D System, USA), MMP-9 (1:1,000, Proteintech, United States), c-Myc (1:1,000, Proteintech, United States), total β-catenin (1:1,000, CST, United States), and pSer675-β-catenin (1:1,000, CST, United States).

2.11 Immunofluorescence assay

Cells on coverslips were washed twice with PBS buffer and fixed in 4% paraformaldehyde for 15 min at room temperature. Next, the cells were permeabilized in 0.2% Triton for 5 min, washed three times with PBS, blocked in 1% BSA/PBS at room temperature for 1 h, and incubated with the following primary antibodies (β-catenin, diluted 1:100; pSer675-β-catenin, diluted 1:100) overnight at 4 °C. Subsequently, the cells were washed 3 times with PBS and incubated with secondary antibody (goat-anti-rabbit, iFluor™ 647, HuaBio, China) diluted 1:500 in PBS containing 1% BSA for 1 h at room temperature. After washing with PBS, cell nuclei were stained with 4′,6 diamidino-2-phenylindole (DAPI) (Solarbio, China) for 5 min and then washed 3 times with PBS. Finally, coverslips were viewed under a confocal laser-scanning microscope (FV3000, Olympus, Japan). All immunofluorescence images within each experiment were captured under the same settings.

2.12 In vivo tumor xenograft model

Ten BALB/c nude mice were purchased from Beijing Huafukang Biotechnology Co., LTD (Beijing, China) and randomly divided into two groups. A total of 2 × 106 stable SAA1-overexpressing TE-1 cells or negative control cells were suspended in 0.1 mL sterilized PBS and then subcutaneously implanted into the left armpit of 5-week-old female BALB/c nude mice (5 mice per group). Tumor volume was measured every 3 days. The animals were sacrificed 3 weeks after transplantation, the tumors were removed, and tumor volume and weight were determined. This study was approved by the ethics committee of North Sichuan Medical College (NO. NSMC202181). Animal study experiments were conducted following the laboratory guidelines for animal care.

2.13 Statistical analysis

All data were analyzed using GraphPad Prism 8 software (GraphPad, CA, USA) and expressed as the mean ± standard deviation (SD). Student’s t test was used to evaluate the differences between groups. p values < 0.05 were considered statistically significant. All experiments were repeated independently at least 3 times.