Patients and tumor tissues
A total of 140 human GC patients were enrolled in this study, and 24 GC patients were tested for multidrug resistance by primary culture of GC tissues. All tissues were obtained at the time of surgery between 2016 and 2018 at the First Affiliated Hospital of Chengdu Medical College (Sichuan, China), and detailed clinicopathological parameters were collected. Following excision, all tissues were immediately frozen in liquid nitrogen and stably stored at -80°C until use. All patients gave written informed consent under protocols approved by the institutional review boards at the First Affiliated Hospital of Chengdu Medical College, Chengdu, China.
Cell lines, cell cultures and reagents
The primary human GC cell lines HEK293T and SGC7901 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). The cisplatin-resistant cell subline SGC7901/DDP was generated from the sensitive parental GC cell line SGC7901 in our laboratory. Cells were grown in increasing cisplatin concentrations (the final concentration of DDP was 1 μg/ml) for 4 months. Cisplatin (DDP; Sigma, Inc., St. Louis, MO, USA; 1 μg/ml) was added to the culture medium of appropriate cell sublines to maintain the DDP-resistant phenotype. Cisplatin and sulfasalazine were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Quantitative real-time polymerase chain reaction
Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription reactions were performed using the GoScript Reverse Transcription (RT) System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Real-time PCR was performed using a standard SYBR Green PCR kit (Roche Diagnostics GmbH, Mannheim, BW, Germany) according to the manufacturer’s instructions. GAPDH and U6 were used as the references for lncRNAs and miRNAs. Primers were synthesized by Sangon Biotech (Shanghai, China), and their sequences are listed in Table S1. The 2-ΔΔCt method was used to quantify relative gene expression. Each sample was analyzed in triplicate.
Adenoviral infection of cell lines
An SLC7A11-AS1-overexpression adenoviral vector (Adv-SLC7A11-AS1-GFP) and control adenoviral vector (Adv-GFP) were purchased from GeneChem (Shanghai, China). pHBAd-RNAi-GFP targeting SLC7A11-AS1 and the negative control pHBAd-GFP were designed and synthesized by Hanbio (Shanghai, China). The specific siRNA sequence for SLC7A11-AS1 was 5'-aggtgtctgtgagggtgtttctgta-3'. Cells were seeded at an 80% density in 6-well plates before infection, and the culture medium was changed 8 h after infection. After infection for 48 h, the infected cells were harvested for use.
Wound healing and transwell assays
Cells were cultured in 6-well plates, followed by scratching to create an artificial wound using a 200-μl micropipette tip in the center of each well. Then, the wounded monolayers were washed with phosphate-buffered saline (PBS) to remove cell debris and incubated with serum-free medium. At four time points, the distance between the two edges of the wound was calculated at three different positions.
An invasion assay was performed using a 24-well Transwell chamber coated with Matrigel (BD Pharmingen, San Jose, CA, USA) and separated by polycarbonate membranes with 8-μm pores (Costar, Cambridge, MA, USA). Then, 2.0×103 cells were seeded in the upper chamber with serum-free medium, and the lower chamber was filled with culture medium containing 10% fetal bovine serum. Invaded cells were fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA), stained with 1% crystal violet (Beyotime, Shanghai, China) and quantified. A migration assay was conducted in a similar fashion without Matrigel coating. All experiments were repeated independently in triplicate.
Cell apoptosis
Cells were harvested after adenoviral infection for 48 h, and the cell density was adjusted to 5×105. Then, the cells were washed with precooled phosphate buffer solution (PBS) 2 times and resuspended after the addition of 100 μl buffer solution. According to the manufacturer’s protocol for an Annexin V-APC/7-ADD apoptosis detection kit (KeyGEN BioTECH, China), 5 μl Annexin V-APC and 5 μl 7-amino-actinomycin D (7-ADD) were added to the cultured cells at room temperature for 15 min. The stained cells were then analyzed by using a BD FACS Canto II flow cytometer (BD, USA). The flow cytometry assay was repeated three times.
RNA fluorescence in situ hybridization and immunofluorescence staining
A Cy3-labeled SLC7A11-AS1 probe was generated by RiboBio (Guangzhou, China). Slides were fixed with 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization at 4°C for 5 min. The slides were hybridized overnight with the SLC7A11-AS1 probe and then washed three times with a graded saline sodium citrate solution.
For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 30 min, stained with a rabbit anti-xCT antibody (1:100 dilution; Proteintech, China) overnight, washed with PBS three times, incubated with a Cy3-goat anti-rabbit IgG (H+L) secondary antibody (1:50 dilution; Beyotime, China) and washed with PBS three times. The nuclei were counterstained with DAPI. Fluorescence images were acquired with a confocal microscope (Leica, Wetzlar, Germany).
Immunohistochemistry
Tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Briefly, paraffin sections were dewaxed in xylene and dehydrated through a graded series of alcohol solutions. The sections were then treated with protein blockers and stained with hematoxylin and eosin or incubated with an anti-xCT antibody (1:200; Abcam, Cambridge, MA, USA) according to standard protocols.
Cell viability assays
A CCK8 assay was used to analyze cisplatin toxicity. A total of 1000 cells were seeded in 96-well plates and cultured for 48 h. Then, a CCK8 solution was added and incubated for one hour. The absorbance at 450 nm was determined for each sample.
Luciferase reporter assay
Wild-type (WT) or mutant (Mut) SLC7A11-AS1 and xCT 3′UTR sequences containing the miR-33a-5p binding site were inserted into the psiCHECK-2 vector (Promega, Madison, WI, USA) to generate psiCHECK-SLC7A11-AS1-WT, psiCHECK-SLC7A11-AS1-Mut, psiCHECK-xCT-WT and psiCHECK-xCT-Mut plasmids. Then, the luciferase plasmids (0.16 μg) and miR-33a-5p mimics/mimics NC (5 pmol) were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activities were quantified sequentially by the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions after 48 hours of transfection. The relative firefly luciferase activity was normalized to the Renilla luciferase activity.
Western Blotting
Total protein was extracted, and the concentration was determined using a BCA protein assay kit (Beyotime, China). Protein samples were electrophoresed by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with primary antibodies against xCT, ASK1, p-P38, P38, p-JNK, JNK and GAPDH (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.
Cellular glutathione and ROS production detection
Cellular glutathione levels were measured by a GSH-Glo™ glutathione assay kit (Promega, Madison, WI, USA). After different treatments, cells were incubated in 100 μl mixed GSH-Glo™ Reagent for 30 min, and then 100 μl reconstituted Luciferin Detection Reagent was added for 15 min. Luminescence signals were detected using a Fluoroskan luminescence scanner (Thermo Fisher, Rockford, IL, USA).
Cellular ROS levels were measured by the Reactive Oxygen Species Assay Kit (Beyotime, China). Cells were incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and the supernatants of differently treated cell lysates for 30 min at room temperature. The fluorescence intensity of the cells was analyzed by FCM (BD FACSCalibur; BD Biosciences).
Mouse xenograft study
BALB/c-nude mice (4 weeks old) were randomly divided into three groups (n = 6 per group). SGC7901/DDP cells (5×106) infected with OE-SLC7A11-AS1, OE-NC or a control vector were subcutaneously implanted in the right flank of BALB/c-nude mice. For a chemoresistance study, sulfasalazine (500 mg/kg) was administered every 3 days or cisplatin (2 mg/kg) was administered every 5 days by intraperitoneal injection beginning when the tumor volume reached 100 mm3. Tumor volumes (V = a×b2/2) were measured every 3 days. In addition, tumor weights were determined after 6 weeks. All animal experimental procedures were approved by the Animal Care and Use Ethics Committee of the First Affiliated Hospital of Chengdu Medical College.
Statistical analyses
All statistical data were analyzed with GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA). Differences between groups were analyzed using Student’s t-test or two-way ANOVA. Data are presented as the mean ± standard deviation (SD). All the tests were two-tailed, and P < 0.05 was considered statistically significant.