Patients and clinical samples
Twenty-one pairs of fresh gastric adenocarcinoma (confirmed by preoperative biopsy and postoperative pathology) and adjacent normal tissues were collected from patients who undergone gastrectomy in Chinese PLA General Hospital from January 2020 to December 2020. Samples were collected immediately after tumor resection and kept in liquid nitrogen and then transferred to −80 °C refrigerator for preservation. All patients did not receive any adjuvant therapy (such as neoadjuvant chemoradiotherapy) before surgery. Written informed consents were obtained from all patients. This study was approved by the Medical Ethics Committee of Chines PLA General Hospital in accordance with the Declaration of Helsinki.
Cell lines and cell culture
Human gastric cancer cell lines HGC-27, MKN-28, SGC-7901, MGC-803, BGC-823, NCI-N87 and immortalized gastric epithelial cell line GES-1 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Adherent cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, NY, USA) with 10% fetal bovine serum (FBS) and 1% P/S in a standard condition at 37 °C with 5% CO2. Gastric cancer stem cells (GCSCs) were obtained and cultured as previously described [13, 14]. Briefly, GCSCs were extracted from fresh GC tissues resected in Chinese PLA General Hospital (PLAGH) and cultured in ultra-low-attachment 6-well plates (Corning, NY, USA) with modified DMEM/F12, 2% B27 supplements (Invitrogen, CA, USA), 1% ITS (insulin–transferrin–selenous acid, Corning, NY, USA), 20 ng/ml epidermal growth factor (Peprotech, Hartford, CT, USA), 10 ng/ml basic fibroblast growth factor (Peprotech), 10 ng/ml LIF (Peprotech) and 10 ng/ml gastrin I (Peprotech).
RNA isolation and qRT-PCR
Total RNA from cell lines or tissues was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). RNA quality and concentrations were determined by Nanodrop 2000 spectrophotometer (ThermoFisher, MA, USA). For mRNA quantification, cDNA was synthesized using a NovoScriptTM All-in-one 1st strand cDNA synthesis SuperMix with gDNA Purge, and qRT-PCR was performed using SYBR™ qPCR SuperMix Plus (Novoprotein, Shanghai, China). For miRNA, the Mir-X miRNA First-Strand Synthesis Kit and qRT-PCR TB Green Kit (Takara, Dalian, China) were used for quantification. All the primers used were listed in Additional file 1: Table S1. The expression levels of mRNA and miRNA were normalized to the expression of GAPDH and U6, respectively. The results were analyzed by the 2−∆∆CT method and each sample was analyzed in triplicate.
Oligonucleotides, plasmids and cell transfection
The miRNA mimics, inhibitors and negative controls were purchased from GenePharma (Shanghai, China). Gain or loss of function of miRNAs were accomplished by transfecting corresponding miRNA mimics or inhibitors, respectively. ΔN-GLI2 cDNA was subcloned into pCMV5 vector with endonucleases. ΔN-GLI2 mRNA, a truncated GLI2 version of GLI2 mRNA (the first 984 bases were deleted), can be translated into ΔN-GLI2 which is more transcriptionally active than full length GLI2 [15]. The GLI2 shRNA plasmid (target sequence: GCAACAAAGCCTTCTCCAA) was synthesized by GeneChem (Shanghai, China). The wild-type GLI2 3ʹ-UTR reporter plasmid containing miRNA binding sites and corresponding mutant-type reporter plasmid were purchased from Promega (#E2920, USA). The mimics, inhibitors and plasmids were transfected into cells by Lipofectamine 2000 reagent (Invitrogen). All the experiments were carried out accordance to the manufacturer’s instructions.
Protein extraction and western blot
Cells were lysed on ice for 20 min using RIPA extraction buffer (Beyotime, Shanghai, China) containing protease inhibitor. Proteins were collected and the concentrations were determined by BCA protein kit (Solarbio, Beijing, China). Then, protein lysates were separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Darmstadt, Germany). The membranes were blocked with 5% defatted milk for 1 h and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. The β-Actin expression was used as internal control for normalization. The brands were detected by enhanced ECL chemiluminescence reagent (Beyotime). Details of the antibodies used in experiments were listed in Additional file 1: Table S2.
Cell proliferation assay
The treated and control HGC-27 and SGC-7901 cells were examined by cell counting kit 8 (CCK-8) (Beyotime) to determined cell proliferation. Briefly, cells in logarithmic phase of each group were collected, resuspended, counted and then seeded at a density of 5000 cells per well into 96-well pates. Three wells were assigned for each group. CCK-8 assays were performed at 0, 24, 48, 72 and 96 h after cell culturation. That is, 10 μl CCK-8 reagent (Solarbio) was added to each well and absorbance at 450 nm was measured after the cells were incubated for 1 h at 37 °C. The proliferation curves were plotted to indicate cell viability.
Colone formation assay
Cells of treatment and control groups were harvested and counted respectively. A total of 1000 cells were seeded per well in 6-well plates with complete culture medium. Cells were cultured at 37℃ with 5% CO2 for 10–14 days until visible colonies formed. Cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet for 30 min. Colonies were photographed and counted.
Cell cycle and flow cytometry analysis
Cells cultured in 6-well plates were trypsined, collected, centrifuged and the supernatant was removed. Then cells were washed with ice cold PBS and resuspended in 1 mL PBS. For the cell cycle, cells were fixed in 70% ethanol at 4 °C for 12–24 h. Subsequently, the cells were incubated with propidium staining solution with RNase A (Beyotime) at 37 °C for 30 min. The cell cycles were detected on BD flow cytometer (BD Biosciences, NJ, USA). For CD44 expression, cells were suspended in 80 μl binding buffer (Beyotime) and then incubated with 2 μl PE-Cy7 CD44 primary antibody (#560533, BD Biosciences) at 4 °C for 15–20 min in the dark. After centrifugation, the cells were resuspended with 500ul Hank’s Balanced Salt Solution (HBSS) and the expression of CD44 was detected on BD flow cytometer. Data analysis was carried out by FlowJo software (Version 10, Tree Star, USA).
Transwell assay
The cell migration and invasion experiments were performed using 8 μm pore size transwell chambers (Corning) in 24-well plates. Cell medium containing 10% FBS was added to 24-well plates 0.6 ml per well. The 200 μl serum-free medium containing certain number of cells were added into each upper chamber. For migration, 4–5 × 104 cells were diluted in 200 μl serum-free medium and added to each chamber. For invasion, the chambers were pre-coated with 50 μl diluted Matrigel (Corning, Matrigel: medium = 1:8), and then 8–10 × 104 cells were diluted in 200 μl serum-free medium and added to each chamber. After incubation at 37 °C for 24 h, cells on the upper surface of chamber were swept by cotton swab slightly. Cells on the bottom of the chamber were fixed with paraformaldehyde for 15 min and stained with crystal violet for 30 min. Cells penetrated were imaged and counted at five random fields.
Dual-luciferase reporter assay
Human embryonic kidney cell HEK293T was used for luciferase reporter assay. Once the cultured cells converged to 40–60%, 100 nM miR-144-3p mimics or negative controls (mimic-NC) were co-transfected with wild type (WT) or mutant type (MUT) GLI2 3’-UTR luciferase reporter plasmids respectively. After transfection for 48 h, a dual luciferase assay system (#E2920, Promega, WI, USA) was applied to determined relative luciferase activities according to the manufacturer’s instruction. The firefly luciferase activity was normalized to Renilla luciferase activity.
Immunohistochemistry (IHC)
The paraffin-embedded tissues were cut into sections of 5 μm in thickness, then the sections were dewaxed and rehydrated. The slides were immersed in EDTA buffer (pH = 8.0) and heated in microwave oven with a specific temperature program for antigen retrieval. After rinsing with PBS, the slides were blocked with 3% BSA solution. Then the slides were incubated with primary antibody at 4 °C over night. Next, slides were incubated with HRP-coupled secondary antibodies at room temperature for 1 h and then 3,3-diaminobenzidine (DAB) was used for visualization of peroxidase reaction. Finally, the slides were re-stained with hematoxylin and photographed under microscope, and the images were analyzed.
In-vivo xenograft assay
Five weeks old male BALB/c nude mice were purchased from Vital River Laboratory Animal Co. (Beijing, China) and raised in Specific Pathogen Free condition. Ten nude mice were randomly divided into negative control (NC) group or miR-144-3p mimic group (5 mice per group). Then, 1 × 106 HGC-27 cells transfected with NC or mimics mixed with Matrigel (Corning) at a 1: 1 ratio. The xenograft model was established by injecting 200 μl abovementioned mixture into the right flanks of the nude mice subcutaneously. Tumor sizes were measured and recorded every 3–4 days with a vernier caliper. Three weeks later, the mice were euthanized. Tumors were dissected and measured. Tumor volume was calculated: V = 1/2×length×width2. All the animal experiments were performed in accordance with the guidelines approved by the Laboratory Animal Ethics Committee of Chinese PLA General Hospital.
Bioinformatics analysis
GLI2-targeted miRNAs were predicted by online database TargetScan (http://www.targetscan.org/vert_72/), miRDB (http://mirdb.org/), miRanda (http://www.microrna.org/) and Starbase (http://starbase.sysu.edu.cn/index.php), respectively. The Venn diagram (https://bioinfogp.cnb.csic.es/tools/venny/) was used to intersect the predicted results. TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) and GEPIA (http://gepia.cancer-pku.cn/) database were used to analyze the GLI2 expression level in gastric cancer and adjacent normal tissues. The online database Kaplan–Meier plotter (http://kmplot.com/analysis/index.php) was applied to analyze survival outcomes. The expression of miRNAs and its correlation with target genes were analyzed based on TCGA database.
Statistical analysis
SPSS Version 25.0 (IBM Corp, Armonk, NY, USA) was used for statistical analysis in this study. The results are expressed as mean ± standard deviation (SD). Student’s t-test (two-tailed) or analysis of variance (ANOVA) were applied to two or more groups comparison. The correlation between two parameters was examined by Pearson correlation test. GraphPad Prism 8 and ggplot2 package were used for data visualization. *p < 0.05, **p < 0.01, ***p < 0.001, ns = no significance. P < 0.05 was considered statistically significant.
