Clinical data analysis
A total of 480 colon cancer cases and 41 cases of adjacent tissues in The Cancer Genome Atlas (TCGA) database were used to analyse genes that are under-expressed in colon cancer and metastatic tumors, which were processed by an online analysis system (https://www.xiantao.love/). In addition, 352 genes suggesting a good prognosis for colon cancer were obtained in the Human Protein Atlas database (http://www.proteinatlas.org/). Then, the core tumor suppressor genes indicating good prognosis and with low expression in metastatic colon cancer were analysed by Venn analysis (http://bioinformatics.psb.ugent.be/webtools/Venn/).
G4-CSSD design and identification
We have designed three CSSDs. Control CSSD with scramble DNA (5’-AATTCAAAGAATTAACCTTAATTGAAGAAACAGUACUUUUGUGUAGUACAAAAACTTCAATTAAGGTTAATTCTTTG-3’), CSSD590: miR-590-3p targeting CSSD without G4 loop (5’-AATTCAAAGAATTAACCTTAATTGAAGATTACTAGCTTATACATAAAATTAAACTTCAATTAAGGTTAATTCTTTG-3’) and G4-CSSD590: miR-590-3p targeting CSSD with G4 loop (5’-AATTCAAAGAATTAACCTTAATTGAAGGGGAGGGTTACTAGCTTATACATAAAATTAAGGGAGGGCTTCAATTAAGGTTAATTCTTTG-3’). All the single-stranded CSSD DNAs were synthesised by Genewiz (Beijing, China). After annealing, two DNA loops with an EcoRI site were treated with T4 DNA ligase (Takara, Beijing, China) to form a completely closed circular DNA. Both CSSD590 and G4-CSSD590 circular DNA contained two miR-590-3p binding sequences. Next, G4-CSSD590 circular DNA was annealed in a buffer of 100 mM KCl and 40% PEG200 to form circular DNA with G4 secondary DNA structure. The harvested DNA was separated and identified by native gel electrophoresis. G4 probe IZCM-7 was donated by Prof. Jia-Heng Tan, and the identification of G4-CSSD was carried out in accordance with his experimental procedure(12).
Cell culture and transfection
HCT116 and Caco-2 colon cancer cells were obtained from the Cell Resource Center of the Chinese Academy of Medical Sciences. All cells were maintained in Minimum Essential Medium (MEM, Gibco, USA) containing 10% newborn foetal calf serum (KeyGENE, China). The full-length coding sequences of CLCA1, BGNT6 and UGT2A3 were synthesised (Genwiz, China) and cloned into a pcDNA3.1 vector. The cells were grown in a petri dish for 24 h before transfection. Lipofectamine 3000 (Thermo Fisher, USA) was used to transfect the transgenic plasmid and G4-CSSD into the cells. After 48 h, the morphological changes in the cells were detected, and the RNA and protein levels were extracted for analysis.
Wound healing assay
The transfected HCT-116 and Caco-2 cells were seeded into a 24-well plate at 3.5☓105 cells per well. When the cell density reached 90%, the pipette tip was used to make a straight scratch. After 48 h, the migration distance was recorded under a microscope (Nikon, Japan), and the migration rate was calculated as followed: Test (0h-48h)/Ctrl (0h-48h).
The treated HCT-116 and Caco-2 cells were resuspended in serum-free medium and seeded into the Matrigel-coated upper chamber (Corning, USA) at a final concentration of 1☓105 per well. The complete medium containing 10% foetal bovine serum was added to the lower chamber. After 24 h, the unpassed cells were wiped off with a cotton swab, and the passed ones were fixed with 4% paraformaldehyde for 30 min. After staining with 0.1% crystal violet for 10 min and then washing with running water for 2 min, the invaded cells were photographed under a microscope (Nikon, Japan).
Scanning electron microscope
HCT-116 and Caco-2 cells were seeded on a slide in 24-well plates at a final concentration of 2☓105 per well. Transfection was performed after 24 h. After 48 h, the treated cells were fixed with 2.5% glutaraldehyde at 4 °C overnight. The fixed cells were treated with different concentrations of ethanol (30% to 100%) and tert-butanol (30% to 100%) and placed overnight at 4 °C. After removing the tert-butanol under vacuum and gold spraying, a scanning electron microscope (JEOL, Japan) was used to detect the cell morphology.
Colony formation assay
Transfected HCT-116 and Caco-2 cells were seeded into a six-well plate at a density of 2000 cells per well. The cells were maintained in MEM containing 2% foetal bovine serum and continuously cultured for 2 weeks. The medium was changed every 3 days. When clones appeared, the medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min. Then, the clones were stained with 0.1% crystal violet for 10 min and counted.
HCT116 and Caco-2 cells were seeded into six-well plates at a density of 106 cells per well. After being cultured in a CO2 incubator at 37 °C, the cells were transfected and continuously cultured for 48 h. After digestion with trypsin without ethylenediaminetetraacetic acid, 200 µl binding buffer was added to 105 cells. Then, 5 µl Annexin V-fluorescein isothiocyanate and 10 µl propidium iodide were added, and the cells were incubated at room temperature in the dark for 20 min. The cell apoptosis rate was analysed and measured with a flow cytometer (Millipore, USA).
miRNA quantitative reverse-transcription–polymerase chain reaction (PCR)
To detect the expression of miR-590-3p, we used Trizol (Beyotime, China) to extract the total RNA at 48 h after transfection. A total of 5 µg RNA was used as a template, and miRNA-specific reverse-transcription primers were used to perform reverse-transcription reaction to obtain the template cDNA. Then, PCR was performed using the SYBR Green qPCR Mix kit (Beyotime, China) in accordance with the manufacturer’s instructions to detect the relative expression of miR-590-3p. U6 snRNA was used as the loading control.
Cells were transfected with Lipofectamine 3000 (Thermo Fisher, USA) in accordance with the manufacturer’s instructions and then cultured for 48 h. After discarding the culture medium and washing with 1×phosphate-buffered saline. Total cell protein was extracted with radioimmunoprecipitation assay lysate containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40 and 0.5% sodium deoxycholate. The quantified protein was separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking in Tris Buffered Saline with Tween® 20 (TBST) containing 5% BSA for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies (UGT2A3 polyclonal antibody, Thermo Fisher, 1:500; B3GNT6 polyclonal antibody, Proteintech, 1:500; CLCA1 polyclonal antibody, Affinity, 1:500) overnight at 4 °C . After washing with TBST for three times, the membranes were incubated with horseradish peroxidase (HRP)-labelled secondary antibody for 1 h at room temperature. Then, an ECL chemiluminescence kit (Beyotime, China) was used to visualise the band and detect the protein expression level in the imager (Biolight, China).
Ten 5-week-old BALB/c nude mice were purchased from (Sipeifu, China). The mice were randomly divided into two groups. HCT-116 cells were inoculated subcutaneously at an amount of 107 per mouse. G4-CSSD was injected into the tumor 7 days after the inoculation when the tumor diameter reached 5 mm. G4-CSSD was injected once a week, and the tumor size was recorded every 3 days. The mice were euthanised by CO2 asphyxiation 34 days after the inoculation. The stripped solid tumors were fixed with formalin and used for immunohistochemical staining. For liver metastasis of colon cancer, HCT-116 cells were inoculated into the spleen of BALB/c nude mice at a concentration of 106. After 4 weeks, the mice were euthanised by CO2 asphyxiation, and liver metastasis was examined. For evaluating the role of G4-CSSD in colon cancer liver metastasis. After the mice were anesthetized, an incision of about 1 cm was made on the left side of the abdominal cavity and the spleen was isolated. 2 x 106 HCT-116 cells were injected into the spleen. Experimental mice were administered CSSD 20 μg/mouse every three days. After 5 weeks, the mice were euthanized to observe the liver metastasis. Animal studies were carried out in accordance with the Animal Use Guidelines of National Institutes for the Use of Laboratory Animals. All procedures were reviewed and approved by Animal Ethics Committee of Tianjin International Joint Academy of Biotechnology and Medicine.
The solid tumor specimens were embedded and cut into 4 µm-thick sections. After deparaffinisation and washing with gradient ethanol for rehydration, the slices were placed in 3% H2O2 for 30 min to block endogenous peroxidase. The slides were then microwave boiled in citrate buffer (10 mM; pH 6.0) for antigen retrieval. Then, the sections were incubated with the following primary antibodies overnight at 4 °C: UGT2A3 (1:100), B3GNT6 (1:200) and CLCA1 (1:100). The sections were then incubated with HRP-labelled secondary antibody for 1 h at room temperature. After washing, 3,3-diaminobenzidine staining solution (Beyotime, China) was added for 30 min. Finally, the sections were counterstained with haematoxylin and photographed under a microscope (Olympus, Japan).
All statistical analyses were performed using GraphPad 7.0 (version 7, GraphPad Software, Inc., La Jolla, CA, USA). Two-tailed unpaired Student’s t-test was used to compare the two groups of data when normally distributed. For comparisons between multiple groups, we used analysis of variance with Bonferroni’s multiple comparison analysis. Asterisks indicate significance: *, p<0.05; **, p<0.01; ***, p<0.001