Bioinformatics analysis
GSE131969 was downloaded from GEO DataSets for screening the upregulated circRNAs using limma 3.26.8 with adj.P value <0.05 and log fold change (logFC) >1.5. GSE33810 downloaded from GEO DataSets was used to screen the upregulated differentially expressed genes (DEGs) with P value <0.05 and logFC >1.5, and GSE20347 also downloaded from GEO DataSets was used to screen the upregulated DEGs with adj.P. value <0.05 and logFC >1.5. STRING was performed to analysis the key biological processes for DEGs. TargetScan and circInteractome were applied to predict the miRNAs binding to TXNRD1 and circ0120816, respectively.
Patients collection
36 patients from Wuhan Asia Heart Hospital Affiliated to Wuhan University of Science and Technology have been diagnosed with ESCC. ESCC tissues and corresponding healthy adjacent tissues were used to demonstrate our assumption. The collection and usage of tissue samples according to the ethical standards set out in the Helsinki Declaration. The clinical characteristics of 36 patients were list in Table 1.
RNA extraction, reverse transcription and real-time quantification PCR
Total RNA was dissociated by TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan), and RNA reverse transcription was performed by PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). The 7500 fast instrument (ABI, USA) was used to measure the expression of circ0139153, circ0096710, circ0095414, circ0085539, circ0120816, miR-1305 and TXNRD1 in ESCC tissues or ESCC cells using SYBR Green PCR kit (Takara, Japan). GAPDH was the reference gene for circRNA and mRNA, while U6 was the reference miRNA for miRNA. All the primers used in this study (Table 2) were designed and synthesized from Tiangen Biochemical Technology (Beijing, China).
Cell culture
The human ESCC cell lines (KYSE30, KYSE180, KYSE450 and KYSE510) and the normal esophageal epithelial cell line Het-1A were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Germany). ESCC cell lines were cultured in RPMI 1640 which had been supplemented with 10% FBS and 1% streptomycin/penicillin in a humidified incubator with 5% CO2 at 37 °C.
Cell transfection
Si-circ0120816, miR-1305 mimic, miR-1305 inhibitor, si-TXNRD1 and their negative control (NC) were designed by GeneCopoeia (Guangzhou, China). Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) was used to conduct cell transfection. In brief, cell transfection was carried out at the point that KYSE450 or KYSE510 cells grew into a density of 70%-80%. As described in the protocol, 50nM Si-circ0120816, miR-1305 mimic, miR-1305 inhibitor, si-TXNRD1 or NC was transfected KYSE450 and KYSE510 cells. The transfection efficiency was analyzed by qRT-PCR after 48-h transfection.
RNase R treatment
Total RNA was inactivated by RNase inhibitor (Beijing Tiangen Biochemical Technology, China) at 37 °C for 15 min. Next, equal RNA was used to reverse transcription into cDNA and to analyze the expression of circ0120816 by qRT-PCR which assessed the stability of circ0120816 and its linear isoform.
Nuclear extraction
This performance was carried out by using Nuclear Extraction Kit (Millipore, USA). In brief, 1×107 cells were added warmed trypsin cell detachment buffer for approximately 2 min. Ice cold 1 × Cytoplasmic Lysis Buffer containing 0.5 mM DTT and 1/1000 dilution was added to the sample for 15-min incubation on ice. For nuclear extraction, the nuclear pellet was resuspended in cell pellet volume in ice cold Nuclear Extraction Buffer. A rotator was used to gently agitate the nuclear suspension at 4 °C for 60 min. The nuclear suspension centrifuged at 16,000 × g for 5 min at 4 °C. The supernatant was the nuclear extract. The cytoplasmic fraction or nuclear lysate was used in subsequent RNA extraction.
Luciferase reporter assay
The wild and mutant circ0120816 sequences or wild and mutant TXNRD1 3’UTR sequences synthesized from GeneCopoeia (Guangzhou, China) were subcloned into pmiR-GLO reporter vector (circ-WT, TXNRD1-WT or circ-Mut, TXNRD1-Mut). KYSE450 or KYSE510 cells were seeded in a 96-well plate and then co-transfected with 100 ng of circ-WT or circ-Mut (TXNRD1-WT or TXNRD1-Mut), and 50 nM of miR-1305 mimic or mimic NC. After 48-h co-transfection, Dual-GLO® Luciferase Assay System Kit (Promega, USA) and Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) were used to detect luciferase activity.
RNA immunoprecipitation assay (RIP assay)
RNA immunoprecipitation assay was used to identify the relationship between circ0120816 and miR-1305 by Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) in accordance with the protocols of manufacture. KYSE450 or KYSE510 cells after transfection of miR-1305 were lysed in RIP lysis buffer containing magnetic beads conjugate with human anti-Argonaute2 (Ago2) antibody or negative control IgG. Then, the cell lysates were incubated with Proteinase K and the immunoprecipitation RNA was isolated. Circ0120816 expression was detected by qRT-PCR.
CCK-8 assay
Cell Counting Kit-8 (Vazyme, China) was used to detect cell viability. Briefly, KYSE450 or KYSE510 cells were seeded in a 96-well plate at a count of 1×105 cells/well. At different time points, 10 μL of CCK-8 solution was added to each well of the plate using a repeating pipettor. Then, the plate was incubated in dark for 1 hour. The absorbance at 450 nm was measured using a microplate reader (BioTek, USA).
BrdU assay
The proliferation of ESCC cells was analyzed using 5-bromo-2'-deoxyuridine (BrdU) Kit (YEASEN, China). Cell culture was carried out on 96-well plates with cell density at 1×105 cells/mL according to standard procedures. According to the dilution at a ratio of 1:30, 1 mM BrdU working liquid was prepared and then added into cells (100 μL for each well) for 2-h incubation. Finally, the absorbance at 450 nm was measured in a microplate reader (BioTek, USA).
Cell adhesion assay
KYSE450 or KYSE510 cells were seeded in a 24-well plate. After 48-h transfection, cells were suspended using serum-free culture medium and added into the 96-well plate precoated with collagen I solution at 5×104 cells per well for 1-h incubation at 37 °C. Then, PBS was added to wash away the cells that were not attached. Subsequently, 100 µL 10% ethanol was added to each well for 5-min incubation at 25 °C. The absorbance was determined using a microplate reader (BioTek, USA) at 570 nm.
Caspase 3 activity assay
KYSE450 and KYSE510 cells were seeded in a 96-well plate at 5×104 concentration. Caspase-3 Activity Assay Kit (Cell Signaling, USA) was applied to carry out the detection. In brief, 30 μL cell lysis buffer was added to each well and the culture plate was placed on the ice for 5 min. Then, the lysates were treated by ultrasound on ice and separated by microcentrifugation at 4 °C for 10 min, and the supernatant was transferred to a new tube. 200 µL substrate solution B and 25 µL lysis solution were mixed together on a black culture plate suitable for fluorescence detection. The plate was incubated in the dark. Finally, we read the RFU on the fluorescent plate reader using an excitation wavelength of 380 nm and an emission wavelength of 420 nm.
RNA pull-down
MiR-1305 mimic-biotin and its negative control (Bio-NC) were synthesized from RiboBio (Guangzhou, China). RNA pulldown Kit (BersinBio, China) was used to perform RNA pull-down. Briefly, cell lysis buffer was blended with probe-bead complex. The tubes were put on the magnetic stand for the collection of the beads, and then protein K and DNase A were used to remove protein and DNA, respectively. The RNA separated from RNA-bead complex was reversed transcript to cDNA. Finally, TXNRD1 mRNA expression level was measured by qRT-PCR.
Statistical analyses
We analyzed our results by SPSS 23.0 (SPSS, USA). All results had three experiments independently. The data were shown as mean ± SD. Student’s t-test was used for analyzing differences between two groups, while ANOVA was used for analyzing differences in multiple groups. P <0.05 were regarded as statistically significant.