Thirty-two pairs of primary CRC tissues and noncancerous mucosal tissues were obtained from patients who underwent curative surgery between 2016 and 2018 at the Department of General Surgery, The First Affiliated Hospital of Soochow University (Suzhou, Jiangsu, China). Tissue specimens were stored in a liquid nitrogen tank or formalin tissue fixative immediately after resection. Clinicopathological characteristics, such as sex, age, tumor location, differentiation, growth, stage, lymphovascular invasion, and perineural invasion were obtained from the electronic medical records at the institute. The seventh edition of the American Joint Committee on Cancer guidelines for tumor, node, and metastasis (TNM) classification was used for staging. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Biomedical Research Ethics Committee of the First Affiliated Hospital of Soochow University (Suzhou, China) (2021-NO:213), and the written informed consent was obtained from patients or their families. All clinicopathological characteristics of CRC samples are listed in Table 1.
Cell cultures and transfection
CRC cell lines (HT-29, HCT116, LoVo, SW480, and SW620) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which performs routine cell line authentication using single nucleotide polymorphism and short tandem repeat analyses. All cell lines were passaged for less than 4 months after resuscitation and were cultured in RPMI 1640 medium or DMEM (ThermoFisher Scientific, Carlsbad, California, USA), containing 10% FBS (Gibco), 100 U/mL penicillin G sodium, and 100 µg/mL streptomycin sulfate (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2.
For transfection. PcDNA3.1-Flag-vector and pcDNA3.1- Flag-STING plasmids encoding human wild-type (WT) STING were obtained from the Public Protein/Plasmid Library (Nanjing: China). The plasmid sequences were veriﬁed via Sanger sequencing. After being cultured at 37˚C in a humidified 5% CO2 incubator for 12 h, LoVo cells were transfected with plasmid (50 nM) using Lipofectamine 2000 (Thermo Fisher Scientific: Waltham: MA) according to the manufacturer's protocol. The plasmids were diluted with Opti-MEM (Thermo Fisher Scientific: Waltham: MA) with the concentration of 2ug/ml for transfection and the transfected cells were cultured overnight in medium without antibiotic. Subsequent experiments were performed 24-48 h after plasmids transfection.
Protein extraction and Western blot analysis
RIPA lysis buffer (Sigma Aldrich) was used for 30 min to lyse cells to extract total protein . Total protein was separated by SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5% non-fat milk at room temperature for 1 h. Bands were incubated overnight with polyclonal antibodies (Cell Signaling Technology) and then with a secondary antibody bound to horseradish peroxidase (HRP) at room temperature for 2 h, and then imaged using the chemiluminescence method. Results were quantified using ImageJ software (version: 1.4.3, RRID: SCR_003070) .
Immunohistochemistry (IHC) and immunofluorescence staining
Paraffin-embedded tissues were cut into 5 μm sections. The sections were dewaxed twice in xylene, rehydrated with graded ethanol, and then incubated with 30% hydrogen peroxide for 10 min to block endogenous peroxidase. They were then stained with hematoxylin for 3 min and rinsed with tap water. Next, 0.5% hydrochloric acid ethanol solution was added, the sample was soaked for several seconds and then rinsed with tap water. Subsequently, the sample was stained with eosin solution for 1 min and rinsed with tap water. The samples were dehydrated with ethanol (70%, 80%, 95%, and 100%) and xylene. The tissues were sealed with neutral resin. In addition, the sections were incubated with goat serum for 30 min, then, the goat serum was removed and antibody (Cell Signaling Technology, USA, 1:200 dilution) was added at 4 °C overnight. The slides were washed with distilled water for 10 min three times and incubated with secondary antibody for 30 min. The final staining score was determined according to color intensity and positive cell rates, as described previously . The score of the proportion of positive cells in the immunohistochemical sections (0, <5%, 1, 5–25%, 2, 25–50%, 3, 50–75%, 4, >75%) was multiplied by the score of staining intensity (0, negative, 1, weak, 2, moderate, 3, strong), the staining degree was then categorized into negative (0–1), weakly positive (2–3), positive (4–7), and strongly positive (8–12). In this study, immunoreactive scores of 0–4 were negative and 5–12 were positive. The IHC results were interpreted independently by two senior researchers.
GSE100179 based on platform GPL17586 ([HTA-2_0] Affymetrix Human Transcriptome Array 2.0 [transcript (gene) version] contained 20 paired normal and tumor colorectal samples. We analyzed the difference of gene expression data of 40 samples. And we used “TCGA” of UALCAN (http://ualcan.path.uab.edu/) to analyze the correlation between the mRNA expression of STING in COAD and individual cancer stages and nodal metastasis status. Both GO and KEGG are important bioinformatics tools to annotate genes and research gene functions, biological process and signaling pathway. In our study, we analyzed these genes that interact with STING by Metascape. The PPI network for these genes was predicted using Search Tool for the Retrieval of Interacting Genes (STRING, https://string-db.org/) online database. In addition, we used the "colorectal adenocarcinoma (TCGA, PanCancer Atlas)" dataset to analyze the co-expression of "STING" and "mTOR". CeRNA Network refers to the large-scale regulatory network formed between conding RNA and non-coding RNA. This hypothesis enriches our cognition of diseases and puts forward new ideas for research. At first, we successfully predicted all the miRNAs of STING using TargetScan and ENCORI. Then, we used LncBase v.2 to predicted the lncRNA of these miRNAs (Pr.score>0.8). Meanwhile, we searched the expression and survival analysis of these miRNAs in COAD on ENCORI. At last, ceRNA network was constructed by Cytoscape software. In recent years, tumor regulation by immune cells has become a major topic of discussion. TIMER (Tumor Immune Estimation Resource, https://cistrome.shinyapps.io/timer/) made use of various immune deconvolution methods to calculate the immune infiltrates’ abundances. In this work, we searched “STING, COAD” in “Gene module” and received an analysis of immune infiltration. And we showed the expression data of STING in various cancers and adjacent tissues. All gene expression data were downloaded from UCSC Xena. Then we used R-package “ggpubr” to get all the charts we need.
6-Carboxy-fluorescein phosphoramidate (FAM)-labeled siRNA (siRNA-FAM) uptake
Cells were cultured in 96-well plates for 24 h at a density of 50.000 cells/well. On the day of transfection, the cells were washed once with sterile phosphate-buffered saline (PBS), the transfection reagent (Lipofectamine™) was added into the medium RNAiMAX/liposome ™ 2000) plus 5, 10, or 20 nm FAM-labeled negative control siRNA (FAM siRNA). Then, 100 μL Opti-MEM (1X), GlutaMAX, and 5% fetal bovine serum were used to replace the medium before adding the Lipofectamine RNAiMAX reagent. The reagent and different concentrations of FAM siRNA were diluted 1:1 in serum-free Opti-MEM, incubated at room temperature for 5 min, and then added to each well. After incubation at 37 °C and 5% CO2 for 5 and 24 h, the cells were washed once with PBS. The nuclei were incubated with 1 μg/L Hoechst 33342 fluorescent DNA probe (Cell Signaling Technology) for 15 min. Images were analyzed with ImageJ software, using background subtraction.
SiRNA targeting human STING (specific target sequence:5’-GCAUCAAGGAUCGGGUUU-3’) was synthesized by IBSBIO. A scrambled siRNA (5’-UUCUCCGAACGUGUCACGUTT-3’) was used as a negative control. Cells were seeded in 6-well plates at 2.5-3 × 105 cells/well for 24 h in their respective culture medium before experiments. The STING siRNA and the negative control siRNA (both at 10 nM) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) reagent for CRC cells, as described previously .
Cell viability was measured using a commercial cell counting kit-8 (Donjindo, Japan). Cells were seeded in 96-well plates at a density of 1 × 103 cells/well. After a 24 h incubation, the supernatant from each well was removed and washed twice with warm PBS. The cells in each well were added to 10 μL CCK-8 solution and 90 μL cell culture medium and incubated at 37 °C for another 2 h. Finally, the absorbance at 450 nm was measured for five consecutive days using a multifunctional microplate reader. Each procedure was performed in triplicate.
Transwell invasion and migration assay
Transwell plates (8 mm, 24-well format, Corning Incorporated, USA) were used for cell invasion and migration analysis. The matrix gel used for the invasion test was slowly thawed on ice at 4°C. The chamber insert used for invasion analysis was coated with a dilute matrix gel and dried at 37 °C. Cells (1 × 105) were suspended in 200 μL serum-free medium and placed in the upper chamber of each incubator, 20% FBS containing complete medium was added to the lower chamber as a chemical attractant. After incubation at 37 °C for 24 h, the cells were immobilized with 4% paraformaldehyde. Finally, the transitional cells were stained with 0.3% crystal violet and quantified using a Nikon inverted microscope.
Glucose uptake assay
Cells were seeded into a 24-well plate at a density of 1 × 105 cells/well. After siRNA treatments, cells were exposed to 0.1 mM 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose) (Invitrogen, Waltham, MA, USA) in the culture medium. Plates were incubated at 37 °C with 5% CO2 for a duration as described above. Images were obtained using identical acquisition settings on a fluorescence microscope (Leica). Mean fluorescence intensity was analyzed by Image J.
All data are presented as the mean ± SD. Statistical analysis of the data was performed using GraphPad Prism 8.0 (GraphPad Software, Inc.). The χ2 or Fisher's exact test was used to analyze the associations between categorical clinicopathological variables and STING expression levels. Comparisons among two experimental groups were performed using two‑tailed Student's t‑test. And two‑way ANOVA followed by Sidak's post‑hoc test was used for comparing multiple groups. P<0.05 was considered to indicate a statistically significant difference.