Four human ESCC cell lines (KYSE-30, KYSE-150, KYSE-510, and TE-1) were obtained from the German Resource Center for Biological Material (DSMZ), and 293T cells were purchased from the American Type Culture Collection (USA). The ESCC cell lines were cultured in RPMI1640 (293T cells in DMEM) supplemented with 10% fetal bovine serum (FBS; (#04-001-1ACS, BI), 100 units/mL penicillin, and 100 μg/mL streptomycin and maintained at 37 ℃, 5% CO2.
ESCC organization source
A cohort of 610 subjects with ulcerative ESCCs was recruited between 2008 and 2014 from the Department of Thoracic Surgery, the Affiliated Hospital of Jining Medical University (Shandong, PR China). We collected relevant clinical data and prognostic information of patients. Among them, 470 cases were male and 140 cases were female (3.4:1), with ages ranging within 34–83 years old (mean age = 61 years). A total of 318 patients had long-term follow-up results, and the mean survival time was 29 months (1–95.2 months).
All biopsies were immediately fixed in 4% buffered paraformaldehyde, routinely processed, and embedded with paraffin. Tumors were classified according to standard TNM staging guidelines of UICC (TNM Classification of Malignant Tumours, Eighth edition). The study protocol had been reviewed and approved by the local ethics committee. All patients gave written consent for their tissue samples. This research was approved by the ethics committee of Jining Medical University. Each patient signed an informed consent form.
Representative areas of the ESCC were marked on each hematoxylin–eosin (H&E) slide and tissue paraffin block, and the marked areas of tissue paraffin blocks were sampled for TMAs. TMAs were assembled with a tissue-arraying instrument (Beecher Instruments, Silver Springs, MD, USA) as described by Kallioniemi et al...
In Situ Hybridization
ESCC TMA was dewaxed in xylene, rehydrated in alcohol gradient, and washed two times with DEPC-PBS. The sections were treated with 2 μg/mL proteinase K (Roche) for 15 min at 37 °C and washed three times with DEPC-PBS. Then, the sections were acetylated 15 min at room temperature (acetic anhydride in DEPC-water, 6 N HCl, and triethanolamine) and subsequently washed three times with DEPC-PBS. Sections were prehybridized in hybridization buffer (50% formamide; 5× saline sodium citrate; pH 7.0; 100 μg/mL sheared salmon sperm DNA, 0.5 mg/mL yeast tRNA, and 1× Denhardt’s solution) at 58 °C for 1 h before the buffer was replaced with hybridization solution containing miR probe. The miR-378d detection probes labeled with digoxin at 5′-end was from Boster (#MK10502). Probes were diluted in pre-hybridization buffer to a concentration of 5 nM and hybridized with the sections overnight at 58 °C according to the RNA melting temperature of probes. After hybridization, the sections were washed three times with 2 × SSC and 0.2 × SSC, permeabilized for immunostaining with 0.1% Triton X-100, and washed two times with PBS. Unspecific background was blocked with 5% swine serum diluted in PBS/BSA for 30 min.
All cell lines were cultured in complete medium until the cells reached 90% confluence. Different concentrations of PTX were added to the different cells, which were then treated for 24 h. PTX was then withdrawn, the medium was replaced, and the cells were cultured until no significant cell death was observed.
Sequencing of miRNA and microarray analysis
The small RNA of TE-1 control and TE-1-PTX (9 days) treated with PTX was used for miRNA sequencing. The miRNA-sequencing libraries were constructed according to the protocol for the Illumina small RNA sample preparation kit. Sequencing was performed on an Illumina HiSeq 2000 sequencer. Library construction and sequencing were performed at Genergy Biotech (Shanghai). miRNA expression was analyzed with miRdeep18.104.22.168 ., and differentially expressed miRNAs were identified using an FDR cutoff value of 0.05. mRNA expression profiling was conducted with Roche NimbleGen Human 12 × 135 K Gene Expression Array by KangChen Bio-tech. Raw data were processed with RMA algorithm, and differential expression analysis was performed with R package limma37 (Version 3.22.7).
Transfection of plasmids was performed using LipofectamineTM 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. Transfection of miRNA mimics or inhibitors (Ribobio, China) was performed using Lipofectamine RNAiMAX (Invitrogen, USA) at a final concentration of 20 nM.
Lentivirus packaging and transduction
Vectors were packaged in 293FT cells using ViraPower Mix (Genepharma). After culturing for 48 h, lentiviral particles in the supernatant were harvested and filtered by centrifugation at 500 g for 10 min, and transfected into ESCC cells. The cells were then cultured under puromycin (10 μg/mL) selection for 2 weeks, after which real-time PCR was used to determine the level of miR-378d. Cell lines stably expressing miR-378d-inhibitor or negative control (NC) vector were designated as Lv-miR-378d-inhibitor and Lv-miR-NC cells, respectively.
Cells were lysed in ice-cold RIPA buffer containing a protease-inhibitor cocktail (Roche). Protein content was quantified with a BCA protein assay kit (Thermo Fisher Scientific). About 30 μg of protein was subjected to electrophoresis, transferred onto PVDF membranes (Millipore), and blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. Membranes were incubated overnight with the following primary antibodies: anti-AKT (dilution 1:1000; CST), anti-AKT1 (1:1000; CST), anti-p-AKT(Ser473) (1:1000; CST), anti-p-AKT (Thr308) (1:1000; CST), anti-β-catenin (1:2000; Proteintech), anti-ALDH1A1 (1:1000; Abcam), anti-Vimentin (1:500; CST), anti-MST1 (1:1000; Proteintech), anti-MST2 (1:1000; Proteintech), anti-LATS1 (1:1000; Proteintech), anti-LATS2 (1:1000; Proteintech), anti-p-LATS1/2 (1:1000; Absin), anti-YAP (1:1000; Ptoteintech), anti-p-YAP (1:1000; Absin). Anti-GAPDH antibody (1:3000; Proteintech) and anti-α-tubulin antibody (1:3000; Proteintech) served as endogenous controls. The specific bands were visualized using secondary anti-rabbit or anti-mouse antibody (1:3000; Proteintech), enhanced chemiluminescence detection kit (Millipore), and FluorChem FC2 Multi-Imager II (Alpha Innotech).
Transwell migration and invasion assay
In vitro cell migration assay was performed using transwell chambers (8 mm pore size; Corning). Cells were plated in serum-free medium (2 × 104 cells per transwell). Medium containing 15% FBS in the lower chamber served as a chemoattractant. After 48 h, the nonmigrating cells were removed from the top face of the filters by using cotton swabs, and the migratory cells located on the bottom side of the chamber were stained with crystal violet, air dried, photographed, and counted. Images of five random fields at 10× magnification were captured from each membrane, and the number of migratory cells was counted. Similar inserts coated with Matrigel were used to determine the cellular invasive potential in the invasion assay.
Matrigel 3D cell culture
Cells (5×103/50 μL) were seeded onto 96-well plates with a round-bottom lid made of ultralow attachment polystyrene (#7007, Costar, USA). The cells were cultured overnight and found to form one sphere per well. After discarding the medium and adding 75 μL of melted Matrigel (BD, USA) to resuspend the cell sphere, the mixture was incubated for 30 min for settling. Finally, 200 μL full medium/well was added, and the medium was changed every other day.
Cells were seeded onto six-well plates (5 × 102 cells per plate) and cultured for 10 days. The colonies were stained with 1% crystal violet for 30 s after fixation with 10% formaldehyde for 15 min and then imaged using the camera of an iPhone 5S (Apple, Inc., Cupertino, CA, USA).
Dual-luciferase reporter assay
In a typical procedure, 293T cells (3×104 cells per well) grown on a 24-well plate were co-transfected with luciferase reporter miRGLO-AKT1-3′UTR plasmid (WT or mutation type; Genepharma, Shanghai, China) (200 ng per well) and miR-378d (20 nM) using Lipofectamine™ 3000 (Invitrogen, USA). About 24 h later, a dualluciferase reporter assay kit (Promega, USA) was used to measure the luciferase and renilla activity of these samples according to the manufacturer’s instructions.
F‑actin cytoskeleton fluorescence staining
Cells were grown on laminin-coated glass cover slips, fixed in 4% paraformaldehyde, and stained with Phalloidin (Molecular Probes, USA). Cells were observed using a fluorescence microscope (Leica, Germany).
CCK8 was used to assess cell viability. KYSE510 and TE-1 cells (1 × 104) were seeded onto a 96-well plate in quintuplicate per well. About 12 h later, the cells were incubated with a gradient concentration of therapeutic drugs for 48 h. The medium was removed, RPMI1640 (90 μL) and CCK8 (10 μL) were subsequently added to each well, and the mixture was incubated for 3 h at 37 °C. A microplate reader was used to measure the optical density (OD) at 450 nm. The degree of drug response for tumor cells was estimated by dividing the half-maximal inhibitory concentration (IC50).
IHC analysis was performed on the cell-block sections from the cultured cells by using the following primary antibodies: anti-AKT1 antibody (dilution 1:100; Proteintech), p53 (1:100, Afifinity), anti-YAP (1:100, Proteintech), anti-TAZ (1:100, Proteintech), and β-catenin (1:100, Proteintech).
About 3×104 cells were seeded on the cover glass of sixwell plates and cultured for 48 h. After fixing with 4% paraformaldehyde in a 37 ℃ water bath for 15 min to maintain cell morphology, cells were permeabilized in PBS + 0.3% Triton X-100 for 15 min, and incubated with anti-p53 (1:100, Proteintech), anti-YAP (1:100; Affinity), and anti-TAZ (1:100; Proteintech) at 4 ℃ overnight, followed by anti-rabbit Cy3-labeled secondary antibody (1:150; ABclonal). PBS washing was performed required for each step.
The animal protocol was approved by the ethical review committee of the Affiliated Hospital of Jining Medical University. Eight-week-old male BALB/c nude mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were anesthetized with 4% chloraldehyde hydrate (100 μL/10 g), and body temperature was maintained by heating blankets. The thoracoabdominal skin of nude mice was sterilized with 75% alcohol and iodophor, respectively. After cutting open the skin at the lower right of the cartilago to expose the lobe of the liver, cell suspension (50 μL/25 μL serum-free and 25 μL of Matrigel containing 500 000 cells) was injected into the liver capsule slowly. Then, the syringe was pulled out and the injection port was pressed for 2–5 min with an iodophor cotton ball. Finally, the incision was sutured layer by layer according to the anatomical structure. Mice were sacrificed 44 days after tumor-cell inoculation. Afterwards, liver tissues, lung tissues, and abdominal-metastasis tumors were fixed in 4% saline-buffered formalin, embedded in paraffin, sectioned at 4 μm, and stained with H&E and IHC.
Statistical analyses were performed using the SPSS 13.0 software package (SPSS, Chicago, IL, USA) and GraphPad Prism Software (version 6, La Jolla, CA, USA). For statistical comparison of two groups, two-sided Student’s t test with the same variances was used. Differences between variables were analyzed by two-tailed or Fisher exact tests. Survival curves were plotted using the Kaplan–Meier method and compared with log-rank tests. Multivariate survival analysis was performed for all parameters found to be significant in univariate analysis using a Cox regression model. Comparisons between groups for statistical significance were performed with a two-tailed Student t test. Data are presented as the mean ± SD. P values < 0.05 were considered significant.