Patients, clinical samples and follow-up
A total of 34 pairs of HCC tissues and adjacent non-tumor tissues were obtained from HCC patients from the department of General Surgery of the First Affiliated Hospital of Nanchang University. All of the patients underwent radical resection between October 2017 and January 2018. The fresh specimens were stored in liquid nitrogen. The clinical data was collected from electronic medical record and the survival information was obtained by follow-up. The methods of follow-up were similar to the previous research . All of the patients did not receive any adjuvant before operation and signed the written informed consent. Our study was compliance with the Helsinki Declaration and was approved by the institutional review of The First Affiliated Hospital of Nanchang University.
Cell lines and cultures
The HCC cell lines MHCC-97H, MHCC-97L were obtained from the Liver Cancer Institute of Fudan University (Shanghai, China). The HCC cell lines Huh7, SMMC-7721, HepG2 and the normal liver cell line L02 were obtained from the Chinese Academy of Sciences (Shanghai, China). All of them were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 100U/ml penicillin and 100ug/ml streptomycin (Gibco, USA). All cells were placed in an incubator with 5% CO2 and humidified at 37℃.
RNA extraction and quantitative real-time PCR (RT-qPCR)
Total RNA was obtained from the cultured cells or fresh-frozen tissues by using TRIzol Reagent (Invitrogen, USA) and reverse transcribed into cDNA by using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan) in accordance with the manufacturer’s instructions. RT-qPCR was performed in a StepOnePlusTM Real-Time PCR System (Applied Biosystems, USA) by using TB GreenR Premix Ex TaqTM II (Tli RnaseH Plus) Kit (Takara, Japan). The β-actin was set as an internal control. All data were analyzed following the method of 2-△△CT. The primers used in the current study were as follows: SFN: forward primer: 5’-TGACGACAAGAAGCGCATCAT-3’, reverse primer: 5’- GTAGTGGAAGACGGAAAAGTTCA -3’.
Establishment of overexpressing and knockdown HCC cells
The knockdown and ectopic expression lentiviruses for SFN, and the corresponding control lentivirus were synthesized by HANBIO (Shanghai, China). The two shRNA sequences (names as: shSFN#1 and shSFN#2) and cDNA clone (names as: SFN) are presented in the Supplementary Table 1. The transfection was performed in cell lines according to the manufacturer’s protocol. In brief, 1×105 MHCC-97H or SMMC-7721 cells were seeded in 6-well plates in DMEM with 10% FBS. The cells were transfected when the cell confluence was about 60%. 1 ml fresh medium with 20 µl lentivirus solution replaced the old medium. After 4 h, another 1 ml fresh medium added into each well. The medium was replaced after 24 h. The stable transfected cell lines were selected by puromycin with final concentration of 2 ug/ml. The efficiency of transfection was verified by RT-qPCR and WB.
Cell proliferation assay
The cell proliferation was assayed using Cell Counting Kit-8 (CCK-8; Dojindo, Japan) according to the protocol of manufacturer. Briefly, 5×103 MHCC-97H or SMMC-7721 cells were seeded into 96-well plates when it stable transfected lentivirus past 48 h, 5 wells for per group. Then, at the time of 0, 24, 48, 72, 96 h, each well was added 10 μL CCK-8 reagent, and the cells was incubated in incubator for 2 h. At last, the multimode reader (TECAN SPARK 10M, Switzerland) was used to measure the absorbance at 450 nm.
Colony formation assays
Infected cells (1000 cells/well in SFN over-expressing group and 500 cells/well in SFN knockdown group respectively) were plated in 6-well plates, and then medium was replaced every five days. After two weeks, the cells were fixed by 4% paraformaldehyde and stained by 1% crystal violet. Then, the 6-plates were washed with running water. The number of cell colony was counted on the pictures.
Wound healing assay
After transfected 48 h, MHCC-97H or SMMC-7721 cells were seeded into 6-well plates. When the cells reached to 95% confluency, the scratch wound was got by a 200 μL pipette tip through drawing lines on the surface cells in 6-well plates. The images of wound healing at 0 and 24 h were photographed using microscope (10×) from each well.
Migration and invasion assays (Transwell)
The transwell chambers (Corning, USA) with the pore size at 8 μm and 24-well plates were used to assay the cell migration and invasion assays. For the migration experiments, 5 × 104 cells were seeded into the upper chambers in 200 μL DMED medium without FBS, while 700 μL DMEM containing 20% FBS was added in the bottom plates. For the invasion experiments, 50 μL Matrigel/DMEM (1:8, BD Biosciences, USA) was added into the upper transwell chambers. The other procedures were similar to the migration except for the number of cells (1 × 105 cells). After incubation for 24 h, the chambers were fixed in the 4% paraformaldehyde and stained with 0.1% crystal violet. Then, the cells or Matrigel on the upper chambers were removed by cotton swab. HCC cells were counted from five random fields by using microscope (10 ×).
The xenograft mice models
The 6-week-old male BALB/c nude mice purchased from Hunan SJA Laboratory Animal CO. LTD were used to explore the role of SFN on the tumor growth (six mice for each group). A total of 5 × 106 of HCC cells transfected with lentivirus in 100 ul DMEM medium containing 50% Matrigel were subcutaneously injected into the right upper flank regions of mice. The tumor length (L) and width (W) were measured every three days. The equation 1/2 × L × W2 was used to calculate the tumor volume. After 34 days, the mice were put into the euthanasia box and carbon dioxide was injected into the box at the rate of 30% of the volume of the euthanasia box per minute. After the animals stopped breathing and its pupils dilated, the next experiments could be carried out. The tumors were excised for further study. All animal researches were approved by the Medical Experimental Animal Care Commission of The First Affiliated Hospital of Nanchang University.
Luciferase reporter assay
The reporter plasmids encoding TOP-flash or FOP-flash with TCF/LEF DNA binding sites and control plasmids pTK-RL were purchased from Beyotime Biotechnology (China). Infected cells were plated in 24-well plates, the pTK-RL and the flash plasmids were co-transfected into the cells. After 48h, the luciferase activity was analyzed as normalized to Renilla luciferase activity.
The total proteins from cells and tissues were isolated with RIPA buffer containing protease inhibitors. After centrifugation, the supernatants were collected for subsequently experiments. The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, USA) were used to extract nuclear proteins. The BCA Protein Quantitation kit (Pierce, USA) was used to measure the protein concentration. A total of 30 μg protein from cells or tissues were separated by 8 % - 12 % SDS-PAGE and transferred into the polyvinylidene fluoride (PVDF, Millipore, USA) membranes. The PVDF membranes were blocked with 5 % skimmed milk for 1 h at 37 ℃ and incubated with primary antibodies against SFN (ab193667, 1:1000, Abcam), β-actin (ab8226, 1:1000, Abcam), E-cadherin (#3195, 1:1000, Cell Signaling Technology), N-cadherin(#13116, 1:1000, Cell Signaling Technology), Vimentin(#5741, 1:1000, Cell Signaling Technology), MMP-2 (ab92536, 1:1000, Abcam), MMP-9 (ab76003, 1:1000, Abcam), β-Catenin(#8480, 1:1000, Cell Signaling Technology), Non-phospho (active) β-Catenin(#19807, 1:1000, Cell Signaling Technology), GSK-3β(#12456, 1:1000, Cell Signaling Technology), Phospho-GSK-3β (Ser9) (#5558, 1:1000, Cell Signaling Technology), Axin2(ab109307, 1:1000, Abcam), and c-Myc(ab32072, 1:1000, Abcam) overnight at 4 ℃. After washed with Tris-buffered saline Tween buffer, the membranes were incubated with HPR-rabbit (SA00001-2, 1:5000, proteintech) or HPR-mouse (SA00001-1, 1:5000, proteintech) at 37 ℃ for 1 h. The intensity of band was quantified by ImageJ (National Institutes of Health, USA) and the specific details were described as previously .
SFN expression in TCGA, GEO and Oncomine databases
The gene expression profiles of liver hepatocellular carcinoma (LIHC) obtained from RNA-Seq HTSeq- FPKM platform was downloaded from GDC Data Portal of The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/repository) consisted of 374 HCC tissues and 50 adjacent non-tumor tissues. Limma package (R) was used to identify SFN between tumor and non-tumor tissues.
For Gene Expression Omnibus (GEO) datasets (https://www.ncbi.nlm.nih.gov/geo/), we downloaded ten expression microarray profiles (GSE: 14520, 25097, 45114, 45436, 55092, 57555, 60502, 76427, 77314, 101728) including HCC tissues and non-tumor tissues. Except for the SFN expression of GSE77314 was directly calculated by the XLSX data downloaded from GEO datasets, difference of SFN expression between tumor and non-tumor tissues of others GEO series were performed by Limma package (R).
For Oncomine database (https://www.oncomine.org/), there were four studies compared SFN expression between hepatocellular carcinoma and normal samples. We comprehensively analyzed four studies with following threshold: p-Value ≤ 1E-4, fold change ≥ 2, gene ranks in the top 10%.
Prognosis Analysis based on online database
To investigate the prognosis value of the SFN gene in patients with HCC, we conducted survival analysis and univariate and multivariate analysis. The univariate and multivariate Cox regression analysis of OS were performed by “survival” and “survminer” packages (https://cran.r-project.org/web/packages/) based on R language. The data downloaded from Kaplan-Meier plotter database (http://kmplot.com/analysis/) was used for survival analysis. Graphpad prism software (Version 7) was used to plot all survival curves.
The data statistical analyses in this paper were performed using SPSS 22.0 software and GraphPad Prism 7. Mann–Whitney U-test, student t test or Chi-square test, and Spearman's rank analysis was performed according to the type of variables. A two-tailed P ＜ 0.05 were considered as statistically significant.