Bioinformatics analysis
The GSE131969 downloaded from GEO DataSets was employed to screen the upregulated circRNAs, while GSE33810 and GSE20347 downloaded from GEO DataSets were used to identify the upregulated differentially expressed genes (DEGs). The upregualted circRNAs and the DEGs with adj.P value <0.05 and log fold change (logFC) >1.5 were selected in this study. STRING algorithm was utilized to analyze the key biological processes for DEGs. TargetScan and circInteractome analyses were later carried out to predict the miRNAs that could bind to TXNRD1 and circ0120816, respectively.
Patients collection
A total of 36 patients from Wuhan Asia Heart Hospital Affiliated to Wuhan University of Science and Technology participated in this study. ESCC tissues and corresponding adjacent healthy tissues from these 36 ESCC patients were collected and used to explore the research objectives. Before data collection, informed consent was obtained from all the participants. The collection and usage of tissue samples were performed according to the ethical standards set out in the Helsinki Declaration and approved by the Ethical Committee of Wuhan Asia Heart Hospital Affiliated to Wuhan University of Science and Technology. The clinical characteristics of the 36 patients are shown in Table 1.
RNA extraction, reverse transcription and real-time quantification PCR
The dissociation of the RNAs was performed with the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan), and the RNAs were quantified using NanoDrop 2000 (Thermo Fisher Scientific, USA). After that, 1 mg RNA was reverse-transcribed with the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). The 7500 Fast Dx Real-Time PCR Instrument (ABI, USA) was eventually used to measure the expression of circRNA, miRNA and mRNA in ESCC tissues and cell lines. The tumor tissues of the xenograft mice model were examined with the SYBR Green PCR Kit (Takara, Japan). GAPDH was used as the reference gene for circRNA and mRNA, whereas U6 was utilized as the reference gene for miRNA. Designed and synthesized from Tiangen Biochemical Technology (Beijing, China), all the primers used in this study are shown in Table 2.
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) and the STR profiling have been done for all the cell lines. All the ESCC cell lines were cultured in an RPMI 1640 medium, which had been supplemented with 10% FBS and 1% streptomycin/penicillin in a humidified incubator containing 5% CO2 at 37 °C. The mycoplasmas in the culture cells were tested once in three months.
Cell transfection
The circ0120816 siRNA (Si-circ0120816), circ0120816 shRNA (Sh-circ0120816), circ0120816 overexpression plasmid (OE- circ0120816), miR-1305 mimic, miR-1305 inhibitor, TXNRD1 siRNA (si-TXNRD1) and their negative control (NC) were designed by GeneCopoeia (Guangzhou, China). Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific, USA) was used to conduct cell transfection. KYSE450 or KYSE510 cells at the exponential growth stage were digested with trypsin and seeded in a 6-well plate at a density of 2 ×105 cells/well. The cell transfection process was then carried out when the density of KYSE450 or KYSE510 cells reached 70%-80%. As described in the protocol of Lipofectamine 2000 Transfection Reagent, 50nM Si-circ0120816, miR-1305 mimic, miR-1305 inhibitor, si-TXNRD1 or NC was transfected into KYSE450 and KYSE510 cells. After 48-h transfection, qRT-PCR was used to analyze the transfection efficiency.
RNase R degradation assay
After KYSE450 or KYSE510 cells were extracted, the total RNAs were inactivated with an RNase inhibitor (Beijing Tiangen Biochemical Technology, China) at 37 °C for 15 min. An equal RNA was then used to perform reverse transcription into cDNA, and qRT-PCR was utilized to analyze the expression of circ0120816. This procedure was done to assess the stability of circ0120816 and its linear isoform. Three independent repeats were conducted for each set of the RNase R treatment experiment.
CircRNA subcellular localization assay
The cytoplasmic and nuclear components were first extracted using the Nuclear Extraction Kit (Millipore, USA). The manufacturer’s guideline was followed religiously: 1×107 cells were collected and supplemented with warmed trypsin cell detachment buffer, and the samples were then incubated for 2 min. Ice-cold 1 × Cytoplasmic Lysis Buffer, which contained 0.5 mM DTT, was added to the sample before incubating it on ice for 15 min. The cytoplasmic fraction was collected after 3-min centrifugation at maximum speed. As for the nuclear extraction, the nuclear pellet was resuspended in the cell pellet volume in an ice-cold Nuclear Extraction Buffer. A rotator was subsequently used to agitate the nuclear suspension gently at 4 °C for 60 min. The nuclear suspension centrifuged at 16,000 × g for 5 min at 4 °C, as well as the supernatant, was collected for the nuclear extract. Finally, the cytoplasmic fraction and nuclear extract were subjected to RNA extraction and RT-qPCR with or without RNase R treatment. This extraction was done to determine the subcellular localization of the circular and linear form of circ0120816. Three independent repeats were conducted for each set of the circRNA subcellular localization assay.
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 the pmiR-GLO reporter vector (circ-WT, TXNRD1-WT or circ-Mut, TXNRD1-Mut). After that, 5×103 per well of KYSE450 or KYSE510 cells were seeded in a 96-well plate and incubated for 24 hours. Next, the samples were 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, the cells were collected and lysed. The Dual-GLO® Luciferase Assay System Kit (Promega, USA) and Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) were eventually used to detect luciferase activities. Three independent repeats were conducted for each set of the luciferase reporter assay.
RNA immunoprecipitation assay (RIP assay)
RNA immunoprecipitation assay was performed to identify the relationship between circ0120816 and miR-1305. The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA), which was used in accordance with the user guide, was used to identify the relationship between circ0120816 and miR-1305. 1×106 KYSE450 or KYSE510 cells were lysed in the RIP lysis buffer. The cell lysates were then incubated with magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody or negative control IgG at 4°C for 12 h. After that, the sample was treated with Proteinase K for 30 min at 37°C. Finally, the magnetic beads were washed twice with the RIP buffer, and the immunoprecipitated RNA was isolated with the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan). After RT-qPCR analysis, circ0120816 expression in the immunoprecipitated RNA was detected. Three independent repeats were conducted for each set of the RIP assay.
CCK-8 assay
Cell Counting Kit-8 (Vazyme, China) was used to detect cell viability. KYSE450 or KYSE510 cells were seeded in a 96-well plate at a density of 1×104 cells/well. At different periods (0 h, 24 h, 48 h and 72 h), 10 μL of CCK-8 solution was added to each well of the plate using a repeating pipettor. The plate was incubated in the dark for 1 hour. The absorbance at 450 nm was measured using a microplate reader (BioTek, USA). Three independent repeats were conducted for each set of the CCK-8 assay.
BrdU assay
The proliferation of ESCC cells was analyzed using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology, USA). Cell culture was carried out on 96-well plates with a cell density of 1×104 cells/well in 100 μL culture media. This procedure was performed according to the standard protocol recommended by the manufacturer. The cell wells were later supplemented with 10 μL 10× BrdU labeling solution and then incubated at 37°C for 24 h. Next, the culture media were replaced with the Fixing/Denaturing Solution (100 μL) and then incubated at room temperature for 30 min. Afterward, the plates were washed three times with 1× Wash Buffer and supplemented with 100 μL prepared 1× Detection Antibody solution for 1-hour incubation at room temperature. Subsequently, the plates were washed again, and 100 μL 1× HRP-conjugated secondary antibody solution and 100 μL TMB substrate were in turn added and incubated for 30 min at room temperature. Finally, 100 μL STOP Solution was added to the cell wells, and the absorbance at 450 nm was measured in a microplate reader (BioTek, USA). Three independent repeats were conducted for each set of the BrdU assay.
Cell adhesion assay
Type I collagen (BD Bioscience, CA) was first coated on the 96-well plates. This was done according to the manufacturer's guideline. Then, 50 μL 10 μg/mL type I collagen was added to each well of the plates. After that, the 96-well plates were incubated at 37°C for 1 hour. Next, the transfected KYSE450 or KYSE510 cells were digested with trypsin, suspended in a serum-free culture medium, and seeded in a 96-well plate precoated with collagen I solution at 5×104 cells per well. After 1-h incubation at 37 °C, PBS was added to remove unwanted cells. Subsequently, 100 µL 10% ethanol was added to each well and incubated for 5 min at 25 °C. The absorbance was finally determined using a microplate reader (BioTek, USA) at 570 nm. Three independent repeats were conducted for each set of the cell adhesion assay.
Cell cycle assay
The Propidium Iodide Flow Cytometry Kit (Abcam, USA) was used to analyze the cell cycle of KYSE450 and KYSE510 cells. According to the product protocol, the transfected KYSE450 and KYSE510 cells were first digested with trypsin to obtain single-cell suspension. Then, the cells were fixed with 66% ethanol at 4°C for 2 hours. After that, the samples were washed twice with PBS. After 5-min centrifugation at 500´ g, the cell pellets were obtained and resuspended in prepared 1´ Propidium Iodide + RNase Staining Solution. The samples were then incubated at 37ºC in the dark for 20 min. Finally, the stained cells were loaded to a flow cytometer for flow cytometry analysis. The DNA content represents the cell cycle phase localized by the cells. Three independent repeats were conducted for each set of the cell cycle assay.
Western blot
The total protein in transfected KYSE450 and KYSE510 cells was extracted and quantified with the RIPA buffer (Solarbio, China), which was supplemented with 1% proteinase inhibitor cocktail. The protein concentration was obtained with the Pierce BCA protein assay kit (Thermo Fisher Scientific, USA). Next, the quantified protein was loaded into 10% or 12% SDS-PAGE for electrophoresis separation. The separated protein bands were then electronically transferred to the membranes (Sigma-Aldrich, USA). Following that, the membranes were put under ice for 2 h with 5% BSA at room temperature. This step was followed by the incubation of the membranes overnight with primary antibodies at 4°Cand 1.5-hour incubation with secondary detection antibody (Cat# ab205718, Abcam, USA) at room temperature. Finally, the protein immunoblots were visualized using the Enhanced Chemiluminescent (ECL) Reagent Kit (Thermo Fisher Scientific. USA). The density of the blots was obtained using ImageJ software. All the primary antibodies were purchased from Abcam (USA), including anti-CyclinB1 (Cat# ab32053), anti-ICAM1 (Cat# ab53013), anti-VCAM1 (Cat# ab134047), anti-Cleaved PARP (Cat# ab32561), anti-Bax (Cat# ab32503), anti-Cleaved Caspase-3 (Cat# ab2302), anti-TXNRD1 (Cat# ab124954) and anti-β-actin (Cat# ab8227).
Caspase 3 activity assay
The Caspase-3 Activity Assay Kit (Cell Signaling, USA) was employed to detect the activities of caspase-3 in KYSE450 and KYSE510 cells. In brief, the cells were seeded in a 96-well plate at a density of 5×103/well. After the transfection process, the cells were washed twice with ice-cold PBS. Subsequently, 30 μL cell lysis buffer was added to each well, and the culture plate was placed on the ice for 5 min. The lysates were then treated with ultrasound on ice and separated using microcentrifugation at 4 °C for 10 min. Next, the lysate solution (25 µL) was obtained and mixed with 200 µL substrate solution B on a black culture plate suitable for fluorescence detection. This step was followed by 60-min incubation in the dark. Finally, the absorbance was measured using a microplate at 450nm. Three independent repeats were conducted for each set of the caspase-3 activity assay.
Tumor xenograft mice model construction
SPF-grade BALB/c nude mice at six weeks were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) and housed in a 12/12 h light/dark cycle in a pathogen-free animal facility at Wuhan University of Science and Technology. After 1-week housing, the nude mice were randomly divided into two groups (3 mice in each group). The mice were then subcutaneously injected with 1×106 KYSE450 cells transfected with circ0120816 shRNA. Other mice that were not injected were used as the negative control. At a xenograft period of 1 and 4 weeks, the xenograft mice were intraperitoneally injected with 75 mg/kg luciferin substrate D-luciferin potassium salt (Shanghai, China). The IVIS 200 bioluminescence imaging system (Caliper Life Sciences, Hopkinton, MA) was also used to quantify the fluorescence and bioluminescence marks in the mice. The mice were then euthanized, and the tumor tissues were obtained for further RT-qPCR and histological staining analysis. For the in vivo lung metastasis examination, 1×106 KYSE450 cells transfected with circ0120816 shRNA or negative control were tail intravenously injected into the nude mice (3 mice in each group), respectively. After housing them for 4 weeks, the mice were euthanized, and the lungs tissues of mice were collected for histological staining. All the animal experiments were approved by the Animal Welfare and Research Ethics Committee of Wuhan University of Science and Technology.
Hematoxylin and eosin (H&E) staining
Before H&E staining, the fresh collected mice tissues with 4% polyformaldehyde were fixed for 18 hours. The fixed tissues were subsequently subjected to gradient alcohol dehydration, xylene hyalinization and paraffin embedding. Next, 5 μm-thick tissue sections were obtained by cutting the paraffin-embedded tissues in a rotary microtome. After that, the tissue sections underwent 15-min de-wax with xylene, 30-min hydration with gradient alcohol, and 2-min staining with hematoxylin staining. This step was followed by the treatment of the samples with 1% hydrochloric acid alcohol solution. Finally, the tissue sections were stained with eosin for 10 sec, dehydrated with gradient alcohol and mounted with neutral balsam. The images of stained tissue sections were captured with the microscope camera (Leica, Germany).
RNA pull-down
MiR-1305 mimic-biotin (Bio-miR-1305) and its negative control (Bio-NC) were synthesized from RiboBio (Guangzhou, China). The Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific, USA) was used to perform RNA pull-down. 5×105 KYSE450 and KYSE510 cells were first seeded in the 6-well plates and then kept in an incubator overnight. Then the Bio-miR-1305 or Bio-NC were transfected for 48 h using Lipofectamine 2000 Transfection Reagent. Next, the cell lysis buffer was blended with probe-bead complex and added to the transfected cells for 3-hour incubation at 4°C. After that, the reaction tubes were placed on the magnetic stand for the collection of the beads. Protein K and DNase A were later used to remove the protein and DNA, respectively. Finally, the RNA in RNA-bead complex was eluted using the RNeasy Mini Kit (QIAGEN) and was reversed transcript to cDNA. Finally, the TXNRD1 mRNA expression level was measured using qRT-PCR. Three independent repeats were performed for each set of the RNA pull-down assay.
Statistical analyses
Data analysis was performed with SPSS 23.0 (SPSS, USA) and GraphPad Prism 8.0 (GraphPad Software, USA). All data used for analysis were obtained from three independently repeated experiments. The data were represented in the form of mean ± standard deviation (SD). Student’s t-test was used to analyze the statistical differences between two groups, while ANOVA was employed to examine the statistical differences among multiple groups. The chi-squared test was utilized to obtain the correlation between the expression of circ0120816 and to determine the clinical features of ESCC patients. P-values less than 0.05 were regarded as statistically significant.