Experimental samples
Tissue samples, including ESCC tumor tissues (n=44) and adjacent normal tissues (n=44) from ESCC patients, and blood samples, including ESCC blood (n=26) and normal blood (n=26) from ESCC patients or healthy subjects, were collected from The First Affiliated Hospital of Xiamen. The written informed consent was signed by every participant. The excised tissues were frozen by liquid nitrogen and preserved in -80℃ conditions. The plasma was isolated from blood samples and stored at -80℃ for further detection. This research was approved by the Ethics Committee of The First Affiliated Hospital of Xiamen.
Cell lines
Human ESCC cell lines, including EC9706, KYSE30 and KYSE450, and normal esophageal squamous epithelial cells (HET-1A) were purchased from Bena Culture Collection (Suzhou, China). Another ESCC cell line TE-8 was purchased from SUER Technology Co., Ltd. (Shanghai, China). In line with the instructions, EC9706 and HET-1A cells were maintained in 90% Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco). KYSE30, KYSE450 and TE-8 cells were cultured in 90% Roswell Park Memorial Institute 1640 (RPMI 1640; Gibco) containing 10% FBS (Gibco). All cells were stored at 37℃ conditions containing 5% CO2.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using the Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) from tissues and cells or using GenElute Plasma/Serum RNA Purification Mini Kit (Sigma-Aldrich) from plasma. After the detection of availability, the RNA was reversely transcribed into cDNA using Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) or MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich). Afterwards, KiCqStart SYBR Green qPCR ReadyMix (Sigma-Aldrich) was utilized to conducted qRT-PCR amplification reaction under Bio-Rad CFX96 (Bio-Rad, Hercules, CA, USA). The relative expression was normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or U6 and calculated using the 2-ΔΔCt method. The primers were used in this study, including circ_DLG1, forward: 5’- AAACGAGAGATAAAGGGCTTCT-3’ and reverse: 5’- ACTGCTTTAGAAACTGGGGAGT-3’; DLG1, forward: 5’-gtggatcattca aag cagtgtga-3’ and reverse: 5’-aggctggcaatctcccaagt-3’; miR-338-3p, forward: 5’-ATCCAGTGCGTGTCGTGG-3’ and reverse: 5’-TGCTTCCAGCATCAGTGAT-3’; MAP3K9, forward: 5’-GAGTGCGGCAGGGAC GTAT-3’ and reverse: 5’-CCCCATAGCTCCACACATCAC-3’; GAPDH, forward: 5’- GAGTCCACTGGCGTCTTCAC-3’ and reverse: 5’- ATCTTGAGGCTGTTGTCATACTTCT-3’; U6, forward: 5’- CTCGCTTCGGCAGCACATA-3’ and reverse: 5’-CGCTTCACGAATTTGCGTG-3’.
CircRNA stability detection
RNase R treatment was performed as follows: 2 μg of total RNA was diluted in 10 μl of water with or without 2 U·mg−1 RNase R and 2 μl of enzyme buffer (Epicenter, Madison, WI, USA), then incubated 15 min at 37℃ and then used for qRT-PCR.
Cell transfection
Small interference RNA (siRNA) targeting circ_DLG1 (si-circ_DLG1) (Ribobio, Guangzhou, China) was used for circ_DLG1 knockdown, and siRNA negative control (si-NC) was used as a reference. Circ_DLG1 overexpression (circ_DLG1) was assembled by BersinBio Co., Ltd. (Guangzhou, China) using their commercial expression vector, and empty vector (Vector) served as control. Overexpression vector pcDNA3.1 containing MAP3K9 fragment (Sangon Biotech, Shanghai, China) was used for MAP3K9 overexpression (MAP3K9), empty pcDNA3.1 vector (pcDNA) as the reference. MiR-338-3p mimics or inhibitors (miR-338-3p or anti-miR-338-3p) (Ribobio) were used for miR-338-3p enrichment or inhibition, miR-NC or anti-miR-NC as the respective reference. Lentivirus short-hairpin RNA against circ_DLG1 (sh-circ_DLG1) (Sangon Biotech) was used for circ_DLG1 stable knockdown, sh-NC as the reference. Cell transfection was conducted using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
Cell cycle detection
Cell cycle was detected using a Cell Cycle Analysis kit (Beyotime, Shanghai, China) following the instructions. At 48 h post-transfection, TE-8 and KYSE450 cells were washed twice with phosphate buffered saline (PBS), trypsinized and centrifuged at 1,500 × g for 5 min at 4℃. Then, cells were exposed to 100 µl of RNase A for 30 min at 37℃ and next stained with 400 µl propidium iodide (PI) for 30 min at 4℃ in the dark. Finally, the cells were analyzed using a FACSCalibur system (Beckman Coulter, Brea, CA, USA).
Cell proliferation analysis
After transfection, TE-8 and KYSE450 cells were seeded into 96-well plates at a density of 5 × 103 cells/well and placed at 37℃ containing 5% CO2. Afterwards, 10 µg MTT (Beyotime) was added into each well at the specific time points (0, 1, 2 and 3 d) and incubated for a further 4 h. Next, 50 µl dimethylsulfoxide (DMSO) (Beyotime) was used to dissolve formazan. Eventually, cell proliferation was analyzed by examining the absorbance at 490 nm under a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Cell migration and invasion analysis
For migration assay, TE-8 and KYSE450 cells were seeded into the top of transwell chambers with 8.0-μm pore membranes in 24-well plates (Corning Incorporated, Corning, NY, USA). For invasion assay, however, the chambers were pre-assembled with a thin layer of Matrigel (Corning Incorporated) prior to the cells were seeded. Meanwhile, RPMI 1640 fresh medium containing 10% FBS was added into the bottom of chambers. After incubation for 24 h, the bottom-surfaced cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime) for 20 min. Finally, the cells were photographed and viewed under a microscope (Olympus, Tokyo, Japan).
Western blot
Total protein was extracted using the radioimmunoprecipitation assay (RIPA) buffer (Beyotime) and quantified by BCA assay kit (Beyotime). Then, proteins (20 μg/sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Next, the membranes were suffered blockage buffer and incubated at 4℃ overnight within the primary antibodies against MAP3K9 (ab228752; Abcam, Cambridge, MA. USA), p38 (ab197348; Abcam), phosphor p38 (p-p38; ab4822; Abcam), extracellular signal-regulated kinase 1 and 2 (ERK1/2) (ERKs; ab184699; Abcam), phosphor ERKs (p-ERKs; ab214036; Abcam), Ki67 (ab16667; Abcam) and GAPDH (ab9485; Abcam). Afterwards, the membranes were probed with the horseradish peroxidase (HRP)-labeled secondary antibodies (ab205718; Abcam). The protein blots on the membranes were emerged using the chemiluminescence kit (Beyotime).
Bioinformatics prediction and dual-luciferase reporter assay
The online bioinformatics tool starBase (http://starbase.sysu.edu.cn/) was used to analyze the putative targeted genes.
Wild-type sequences of circ_DLG1 containing the miR-338-3p binding site and mutant-type sequences of circ_DLG1 containing the miR-338-3p mutated binding site were inserted into the pGL4 vector (Promega, Madison, WI, USA), named as circ_DLG1-wt and circ_DLG1-mut. Likewise, wild-type sequences of MAP3K9 containing the miR-338-3p binding site and mutant-type sequences of MAP3K9 containing the miR-338-3p mutated binding site were cloned into the pGL4 vector, named as MAP3K9-wt and MAP3K9-mut. TE-8 and KYSE450 cells seeded into 24-well plates were co-transfected with 50 nM miR-338-3p or miR-NC and circ_DLG1-wt, circ_DLG1-mut, MAP3K9-wt or MAP3K9-mut using Lipofectamine 3000. The luciferase activity was examined using the dual-luciferase reporter assay kit (Promega) after 48-h transfection.
Animal experiments
Animal procedures were conducted conforming to the Animal Care and Use Committee of The First Affiliated Hospital of Xiamen. 5-week-old nude mice (Female, n=8) were purchased from the Animal Core Facility, SIBCB (Shanghai, China). TE-8 cells stably transfected with sh-circ_DLG1 or sh-NC were subcutaneously implanted into the right flank of mouse back. Tumor volume was recorded every 5 d using the formula: length × width2 × 0.5. Tumors were excised after 30 d.
Statistical analysis
Statistical analysis was performed using SPSS 21.0 software (IBM Corp., Armonk, NY, USA). The linear dependence was analyzed by Spearman’s correlation analysis. All data were collected from three independent experiments and displayed as mean ± standard deviation. The Student’s t-test was applied to analyze the differential significance between two groups, and analysis of variance (ANOVA) was applied to analyze significance among more than two groups, followed by the Tukey correction for multiple comparisons. P < 0.05 was considered to be statistically significant.