1.1 Bioinformatics Analysis
Gene and miRNA Expression Quantification data related to ESCA were accessed from TCGA database (https://portal.gdc.cancer.gov/) and “edgeR” package was used to identify the differentially expressed genes (DEGs), with threshold set to |logFC|>1 and padj<0.05. TargetScan (http://www.targetscan.org/vert_72/), miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/index.php) and miRDB (http://www.mirdb.org/) databases were used to predict target mRNAs for the target miRNA, and Venn diagram was plotted to find the target mRNA. Survival analysis on the target mRNA was performed combined with the clinical information obtained from TCGA.
1.2 Cell culture
Normal human esophageal epithelial cell line HET-1A was purchased from Riken BioResource Center (Tsukuba, Japan). Five EC cell lines Eca-109, EC9706, KYSE150, KYSE180 and BIC-1 were purchased from the Cell Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco), streptomycin (100 mg/ml; Gibco) and penicillin (100 units/ml; Gibco), and maintained in an incubator at 37 ℃ with 5% CO2.
- 3 Vector construction and cell transfection
MiR-145-5p mimic, miR-145-5p inhibitor, si-ABRACL and their negative controls purchased from GenePharma (Shanghai, China) were transiently transfected into EC cells by Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) and maintained in corresponding medium under the environment of 5% CO2 at 37 °C. The lentiviral expression vector pLVX-IRES-neo (Clontech) was used to establish ABRACL overexpression vector oe-ABRACL, which was then used to infect cancer cells with the virus particle as the negative control. Mock was the control group only with transfection reagent. The sequences of transfection materials are as follows：miR-145-5p mimic sense: 5’-GUCCAGUUUUCCCAGGAAUCCCU-3’, antisense: 5’-GGAUUCCUGGGAAAACUGGACUU-3’; mimic NC sense: 5’-UUCUCCGAACGUGUCACGUUU-3’, antisense: 5’-ACGUGACACGUUCGGAGAAUU-3’; miR-145-5p inhibitor: 5’-CUUAGCAUCUAAGGGAUUCCUGGG-3’; inhibitor NC: 5’-CGAACUUCACCUCGGCGCGGG-3’; si-ABRACL: 5’-GCGCUCACAGUAGGAGUUU-3’, si-NC: 5’-GCGUUCAACUAGCAGACCA-3’.
1.4 RNA extraction and qRT-PCR
Total RNA was extracted from cells using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) and then reversely transcribed into cDNA by cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative PCR was performed using miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany) under following thermal cycling conditions: pre-denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min (Mei et al., 2017). Primers for miR-145-5p, U6, ABRACL and GAPDH were purchased from GeneCopoeia (GeneCopoeia Inc., Guangzhou, China) and sequenced as below: miR-145-5p forward (stem-loop): 5′-TCGGCAGGGTCCAGTTTTCCCA-3′, reverse: 5'-CTCAAC TGGTGTCGTGGA-3'; U6 forward: 5'-GGAGCGAGATCCCTCCAAAAT-3', reverse: 5'-GGCTGTTGTCATACTTCTCATGG-3'; ABRACL forward: 5′-ACCTCTTTGAAGCATTGGTAGG-3′, reverse: 5′-GCAGCTCTCCTGGATATGTTAC-3′; GAPDH forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′. The 2-ΔΔCt value was used to compare the difference in the relative expression of the target mRNA and miRNA between the control group and the experimental group. The experiment was repeated three times.
1.5 Western Blot
After transfection for 48 h, cells were washed 3 times with cold PBS. Whole cell lysate was added for cell lysate on ice for 10 min and the BCA protein assay kit (Thermo, USA) was applied for determination of protein concentration. A measure of 30 μg of protein samples were processed for separation by polyacrylamide gel electrophoresis (PAGE) at a constant voltage of 80 V for 35 min followed by 120 V for 45 min, and sequentially transferred to polyvinylidene fluoride (PVDF) membranes (Amersham, USA). After being blocked with 5% skim milk for 1 h at room temperature, the membranes were incubated with ABRACL rabbit polyclonal antibody (ab163694, 1:1000, abcam, Cambridge, UK) and GAPDH rabbit polyclonal antibody (ab9485, 1:2500, abcam, Cambridge, UK) overnight at 4 °C. The membranes were washed 3 times with PBST (Phosphate buffered saline containing 0.1% Tween-20) for 10 min each time. Subsequently, the membranes were incubated with horseradish peroxidase (HRP) -labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1:3000, abcam, Cambridge, UK) for 1 h at room temperature. The membranes were washed 3 times with PBST buffer. An optical luminometer (GE, USA) was employed for visualization of protein bands, and the Image Pro Plus 6.0 (Media Cybernetics, USA) software was applied for further analysis.
1.6 Colony formation assay
24 h post transfection, cells were seeded at 5×102 cells/well in 6-well plates. After 10 days, the colonies of the viable cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. Stained colonies were observed and calculated. The experiment was repeated three times.
Transwell migration assay: Cancer cells in logarithmic growth phase were starved for 24 h. On the following day, cells were digested, centrifuged and resuspended to a final concentration of 2×105 cells/ml. A measure of 0.2 ml of cell suspension was added into the Transwell inserts, while 700 μl of pre-cooled RPMI-1640 medium containing 10% FBS was added into the plates. Cells were cultured in an incubator containing 5% CO2 at a temperature of 37 °C. After 24 h, the cells still in the inserts were wiped off with a wet cotton swab, and the cells migrated out of the inserts were fixed using methanol for 30 min and then stained by 0.1% crystal violet for 20 min. Finally, cells were observed under an inverted microscope and five fields of view were randomly selected for cell count.
Transwell invasion assay: 24-well Transwell chambers (8 μm in aperture, BD Biosciences) were used for Transwell invasion assay. Approximately 2×104 cells were plated in the upper chambers that were pre-coated with Matrigel matrix (Corning, Corning, NY). RPMI-1640 medium containing 10% FBS was filled into the lower chambers. The following procedures were as similar as the above migration assay.
1.8 Dual-luciferase reporter gene assay
Eca109 cells were seeded at 6×105 cells/well in 24-well plates and incubated for 24 h. The mutant (MUT) or wild type (WT) 3'-UTR of ABRACL was amplified and then cloned into pmirGLO (Promega, WI, USA) to construct luciferase reporter vectors ABRACL-WT and ABRACL-MUT. Subsequently, ABRACL-WT and ABRACL-MUT were transfected into cells with miR-145-5p mimic or mimic NC by using lipofectamine 2000, respectively. The Renilla luciferase vector pRL-TK (TaKaRa, Dalian, China) was used as a control reporter for normalization. After transfection, cells were cultured in RPMI-1640 medium containing 10% FBS. After 48 h, luciferase activities were measured using Dual-Luciferase Reporter Assay System Kit (Promega Corp., Madison, WI, USA). The experiment was repeated three times.
1.9 Statistical analysis
All data were processed using SPSS 22.0 statistical software, and the measurement data were expressed in the form of mean ± standard deviation. Student’s t test and one-way analysis of variance (ANOVA) were applied for analyzing the comparisons between two groups and among multiple groups. P<0.05 indicates statistically significant difference.