Materials and reagents
The antibodies against SNTB1 (GTX46866) and PCNA (GTX100539) were purchased from GeneTex (San Antonio, TX, USA). The antibodies against PKN2 (35490) and c-Jun (13361) were purchased from Signalway Antibody (College Park, MD, USA). The antibodies against CyclinD1 (2978), CDK4 (12790), Bcl-2 (3498), Bax (2772), Caspase-3 (9662), Caspase-9 (9508), p-ERK1/2 (9102), ERK1/2 (4370), AKT (4691), p-AKT (4060), p-cJun (3270), p-p38 MAPK(9216), p38 MAPK(8690) and GAPDH (2118) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against SNTB1 (PA5-55143), Fetal Bovine Serum (FBS), Trypsin-EDTA (0.25%), Pierce TM BCA Protein Assay kit, and FxCycle PI/RNase Staining Solution was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Annexin-V-AbFluor™ 647 Apoptosis Detection kit and CCK8 kit were obtained from Abbkine (Wuhan, Hubei, China).
Differential expressed genes analysis
In prior work, we screened DEGs on 14 pairs of CRC primary lesions and surrounding non-cancerous tissues (GEO Submission: GSE113513) [11]. Among these DEGs, 8 of DEGs including SNTB1 were selected for further investigation due to the limited amount of published data regarding their involvement in carcinogenesis. In this study, mRNA expression of SNTB1in CRC and control tissues was analyzed thorough TCGA (https://cancergenome.nih.gov/) [12].
Cell lines and culture
Both human CRC cell lines HCT116 and RKO were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT116 cells were cultured in M5′A medium (KeyGEN, Jiangsu, China) and RKO cells were cultured in MEM-alpha medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 1% penicillin-streptomycin (Hyclone, Logan, UT, USA). All cells were cultured at 37°C in a humidified atmosphere with 5% CO2. Short tandem repeat genotyping was performed for examination of mycoplasma contamination of cells using qPCR.
Lentiviral transduction and high-content screening for cell growth
High-content screening (HCS) was performed to assess the growth of CRC cells. Briefly, control shRNA lentivirus and shRNA lentivirus targeting 8 DEGs (the sequences of shRNAs are listed in Supplement Table S1) were constructed by Shanghai GeneChem (Shanghai, China). HCT116 cells were seeded in 12-well plates for 16h prior to lentivirus transduction and then transduced by adding the shRNA lentiviral particles (multiplicity of infection: 10) with GFP into the cell culture medium according to the protocol of the manufacturer. At the end of transduction, 2 x 103 cells in 100 µL of complete medium were reseeded into 96-well plates. Cellomics Array Scan VTI HCS (Thermo Fisher Scientific) was used to monitor the cell growth over 5 days. Image analysis was performed using HCS Studio Cell Analysis Software (Thermo Fisher Scientific).
Quantitative real-time PCR (qPCR) analysis
The total RNA was isolated from cells using RNAiso Plus reagent (Takara, Beijing, China). Reverse transcription into complementary DNA (cDNA) was amplified according to the manufacturer’s instructions using the PrimeScript RT reagent kit (Takara). Tissue cDNA array containing 79 primary CRC and 15 noncancerous colorectal tissues (Cat#: cDNA-hcola095su01) was purchased from Shanghai Outdo Biotech Company (Shanghai, China) and the levels of mRNA encoding SNTB1 and GAPDH were detected using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Premix Ex Tag (Takara). The conditions for qPCR were as follows: pre-denaturation (95℃ for 10minutes), denaturation (95℃ for 15 seconds), annealing and extension (60℃ for 60 seconds) for a total 40 cycles. GAPDH was used as an internal control. Primer sequences are shown in Supplementary Table S2. mRNA levels are presented as: 2ΔΔCt (with Ct being the cycle threshold), where ΔCt [Ct (target gene) - Ct (GAPDH)]. Clinicopathologic features of CRC patients represented in the cDNA array are summarized in Supplement Table S3.
Tissue microarray array
TMA containing 70 pairs of CRC and noncancerous colorectal tissues were obtained from Shanghai Outdo Biotech Company (Shanghai, China; Cat#: HColA180Su15). IHC was conducted to detect the expression of SNTB1 in CRC samples using an antibody against SNTB1 (Rabbit monoclonal to SNTB1; dilution 1:100; Thermo Fisher Scientific), as described previously [13]. Scoring was carried out using a grading system based on staining intensity (no staining, 0; weak, 1; moderate, 2; strong, 3) and percentage of positive-staining cells (1–25% positive, 1; 26–50%, 2;51–75%, 3; 76–100%, 4) [14]. The final score was calculated as intensity score × percentage score. Clinicopathological features of CRC patients are summarized in Supplement Table S4.
Western-blot analysis
Cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors. The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to measure concentrations of total protein. Equal amount of total protein lysate was separated on 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Next the membranes were blocked with 5% skim milk in TBST at room temperature for 2 h and incubated overnight at 4 ℃ with primary antibodies (1:1000), and followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000). Proteins were visualized using an ECL imager (Thermo Fisher Scientific, USA) and band intensities were quantified using ImageLab software. The expression of GAPDH was used as a control. Three independent experiments were performed for each assay.
CCK-8 assay
Transduced cells were re-seeded into 96-well plates (2,000 cells per well) and cultured in at 37°C and 5% CO2 for the indicated time points. Cell Counting Kit-8 reagent (10 ul; Abbkine, Wuhan, Hubei, China) was added to each well, plates were incubated for an additional 2 h at 37°C, and the optical density (OD) was measured at a wavelength of 450 nm. The cell viability was calculated based on the OD for each group.
Colony formation assay
Transduced cells were seeded into 12-well plates at a density of 500 cells per well and cultured at 37°C and 5% CO2 for 10–14 days. Cells were fixed in 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet (Solarbio, Beijing, China) for 20 min at room temperature. Colonies were manually counted. Each assay was performed in triplicate.
Cell cycle and apoptosis analysis
For the cell cycle assay, transduced cells were collected and fixed with 70% ethanol at 4℃ overnight. The fixed cells were centrifuged at 2000 rpm for 3 min and washed, followed by incubation with a mixture of FxCycle PI/RNase Staining Solution (Thermo Fisher Scientific) for 30 min at room temperature. FACS (Fluorescence activated Cell Sorting; Becton Dickinson, CA, USA) was used to analyze cell cycle progression using ModfitLT version 3.0 (Verity Software House). For the apoptosis assay, transduced cells were washed twice with ice-cold PBS and incubated with Annexin-V-AbFlour™ 647 Apoptosis Detection Kit solution (Abbkine, Wuhan, China). The apoptotic rate was analyzed using FACS.
In vivo experiments
A xenograft nude mouse model was constructed to investigate the effects of SNTB1 knockdown on tumor growth. Male BALB/c nude mice (6–8 week old) were obtained from Shanghai Laboratory Animal Center at the Chinese Academy of Sciences and raised in a specific pathogen-free facility at Fujian University of Traditional Chinese Medicine (Fujian, China). All animal procedures were approved by the Committee of Fujian University of Traditional Chinese Medicine (Approval No: FJTCM IACUC 2019050). HCT116 cells or RKO cells were transduced with a lentivirus encoding anti-SNTB1 shRNA (sh-SNTB1) or control shRNA (sh-Ctrl). Cells (1 × 106 in 100 µL PBS containing 50% Matrigel) were injected subcutaneously into the flanks of nude mice (n = 6). Tumor volume was determined every other day using measurements obtained with a vernier caliper and the following formula: 1/2 (larger diameter×smaller diameter2). At the end of the experiment, mice were anesthetized with isoflurane. An IVIS Spectrum live-animal imaging system (PerkinElmer; Santa Clara, CA, USA) was used to capture tumor images. Mice were sacrificed for tissue collection for further use. Signal intensity was quantified as the number of photons within the region of interest per second.
Immunohistochemistry
Tissues were fixed with 4% paraformaldehyde at 4°C overnight, embedded with paraffin, cut into 5µm-thick sections, and mounted onto slides. The slides were dehydrated by graded ethanol. Antigens were retrieved by using microwave heating for 20 min in sodium citrate-hydrochloric acid buffer. Tissue sections were incubated with an antibody against SNTB1 (1:100; Thermo fisher Scientific) or PCNA (1:800; Genetex). Background staining was assessed by omitting the primary antibody. The intensity of staining was evaluated using a scoring system described in detail in the Section “Tissue microarray array”. The overall staining score was calculated by multiplying the intensity score and percentage score together.
TUNEL assay
Apoptotic cells in tissue sections were detected using terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) staining according to the manufacturer’s instruction. The percentage of TUNEL-positive cells and staining intensity were evaluated using a scoring system described in detail in the Section “Tissue microarray array”.
Isobaric tags for relative and absolute quantification (iTRAQ) analysis and protein identification.
iTRAQ was used to identify differential expressed proteins (DEPs) [15, 16]. SDS-PAGE electrophoresis was first carried out for protein quantification. The protein samples were cysteine-blocked and digested, protein labelling and mass spectrometry (MS) analysis were performed. Two-dimensional liquid chromatography-mass spectrometry (2D-LC-MSMS) analysis including reversed-phase chromatographic separation (Agilent Technologies, Santa Clara, CA, USA) and reversed-phase chromatography on a TripleTOF (AB SCIEX, Framingham, MA, USA) was conducted. Proteins were classified as differentially expressed if their expression differed at least 1.5-fold between the two conditions and if the difference was associated with P < 0.05. These proteins were identified using volcano plots and hierarchical clustering plots.
To enrich the biological groups and KEGG pathway, the identified proteins were submitted to Omicsbean (http://www.omicsbean.cn/) software. The significantly enriched gene ontology (GO) categories were reported using a right-sided hypergeometric test, which compares the background set of GO annotations in the whole genome of homo sapiens. The false discovery rate (FDR) was controlled by the Bonferroni step-down test to correct the p-value [17, 18].
To better understand the protein-protein interactions among the differentially expressed proteins of each group, we constructed protein-protein interaction (PPI) networks through Omicsbean. Proteins were then grouped based on their GO annotations with p-value < 0.05 [19].
Statistics analysis
Data were analyzed using SPSS 22.0 software. For survival analysis, SNTB1 mRNA expression in CRC tissues from cDNA array was classified into high or low expression groups based on mean. Kaplan-Meier survival curves were plotted for high- and low-expression groups and the correlation of SNTB1 mRNA expression with overall survival of CRC patients was analyzed using log-rank test. The correlation between SNTB1 mRNA expression and CRC patients’ survival in dataset of COAD sourcing from TCGA was analyzed through Kaplan-Meier Plotter (http://kmplot.com/). Student’s t-test or Mann-Whitney U was used for comparisons between two groups. One-way ANOVA or Kruskal-Walis H was applied to assess multiple group comparisons. All quantitative data are presented as the mean ± SD. P < 0.05 (two sided) was considered statistically significant. All experiments were repeated at least three times.