The expression of SAR1B in CRC was analyzed using GEPIA datasets (http://gepia.cancer-pku.cn/) (14). Moreover, Kaplan-Meier method (14) was used to analyze the association between SAR1B expression levels and overall survival (OS) time in patients with CRC using two public datasets, GSE17536 (15) and GSE17537 (15). In order to explore the potential functions of SAR1B in CRC, the DepMap database was analyzed (https://depmap.org/rnai/index) (16). The function of SAR1B in cancer cell survival was analyzed using DepMap portal (https://depmap.org/rnai/index) based on the results of genome-wide shRNA (or small interfering RNA) inhibition of SAR1B from the Broad (17) and Novartis (18) datasets. A PPI network regulated by SAR1B was constructed using the STRING database (https://string-db.org/cgi/network.pl) (19). The bioinformatics analysis was conducted using the Ingenuity Pathway Analysis system (https://www.nihlibrary.nih.gov/resources/tools/ingenuity-pathways-analysis-ipa).

A total of 15 patients with CRC were recruited for the present study. Patients from Taizhou People's Hospital (Taizhou, China) were pathologically diagnosed with CRC and underwent surgical resection between July 2019 and October 2019. No patient received chemotherapy or radiotherapy treatment prior to surgery. The clinical information of these patients is included in Table I. All patients provided written informed consent, and the present study was approved by the Committees of Taizhou People's Hospital.

CRC cell lines HCT116, SW620 and RKO cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Penicillin (100 U/ml) and streptomycin (100 U/ml) were added to the medium to prevent contamination. Cells were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO₂.

The target DNA sequence (5′-TGGCACAGTACATTGATTA-3′) of SAR1B was selected from the full-length sequence (NM_016103) by Shanghai GeneChem Co., Ltd. According to the sequence of SAR1B, two vectors, shRNA S1 and shRNA S2, were designed. The sequences were as follows: shRNA S1, 5′-CCGGGATGGCACAGTACATTGATTACTCGAGTAATCAATGTACTGTGCCATCTTTTTG-3′; shRNA S2, 5′-AATTCAAAAAGATGGCACAGTACATTGATTACTCGAGTAATCAATGTACTGTGCCATC-3′. The shRNAs were annealed and ligated to the linearized GV115-GFP lentivirus vector (Shanghai GeneChem Co., Ltd.). The plasmid was extracted from the DH5α cells (Shanghai GeneChem Co., Ltd.) and verified by restriction endonuclease digestion followed by Sanger sequencing. The plasmid was extracted and verified by enzymatic digestion and sequencing. RKO cells (ATCC) at a density of 8,000 cells/well were infected with the lentivirus. RKO Cells infected with an empty lentiviral vector were used as a control (shCtrl). Cells at a density of 8,000 cells/well were cultured in RPMI-1640 medium with lentiviruses at a multiplicity of infection of 10 for 24 h at 37°C. A stable SAR1B-knockdown RKO cell line was constructed using the GV115-GFP lentivirus vector system (Shanghai GeneChem Co., Ltd.), which could express GFP. After 72 h of infection, the fluorescence and infection efficiency were determined using an inverted fluorescence microscope at 200× magnification (IX-71; Olympus Corporation). When the infection efficiency was 80%, the expression of SAR1B was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The strong and positive EGFP signals in RKO cells following infection with a lentivirus recombined with shSAR1B or shCtrl indicated that the efficiency of infection in RKO cells and were determined using an inverted fluorescence microscope (magnification, ×200; Olympus).

Total RNA from HCT116, SW620 and RKO cells and 15 paired CRC samples were extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 2 µg RNA RNA samples were reverse transcribed using a Reverse Transcription kit (Roche Diagnostics GmbH) to synthesize cDNA, according to the manufacturer's protocol. The reverse transcription temperature protocol was as follows: 65°C for 10 min, 25°C for 10 min, 50°C for 1 h and 85°C for 5 min. cDNA (1 µl) was used as a template for PCR using the SYBR^® Green qPCR kit (Takara Biotechnology Co., Ltd.), using the ViiA™ 7 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used were as follows: SAR1B, forward 5′-TTTTCCTACGATGGAATCTGGC-3′, reverse 5′-CAGCCTGTCTCCTCGCTTTC-3′; and GAPDH, forward 5′-TGACTTCAACAGCGACACCCA-3′ and reverse 5′-CACCCTGTTGCTGTAGCCAAA-3′. The thermocycling conditions were as follows: Initial denaturation at 95°C for 15 sec, followed by 45 cycles at 95°C for 5 sec and 60°C for 30 sec. The PCR products of SAR1B and GAPDH were 241 and 121 bp, respectively. All samples were analyzed by using the 2^−ΔΔCq method in triplicate (20).

shSAR1B-infected and shCtrl RKO cells were digested with trypsin and resuspended in standard medium after reaching the logarithmic growth phase. Cells were seeded into 6-well plates at a density of 500 cells/well, incubated at 37°C in a 5% CO₂ incubator and observed for 10 days with half of the medium changed every 3 days. Cells were washed with PBS and fixed with 4% paraformaldehyde (1 ml/well; Shanghai Sangong Pharmaceutical Co., Ltd.) for 30–60 min at room temperature. Cells were washed with PBS, stained with 500 µl Giemsa (cat. no. ECM550; Chemicon; Thermo Fisher Scientific, Inc.) for 20 min at room temperature and washed with deionized water three times. Images of cell colonies were captured using inverted light microscope (200× magnification; IX71; Olympus, Tokyo, Japan) and counted using ImageJ software (version 4.0; National Institutes of Health).

An MTT assay (Sigma-Aldrich; Merck KGaA) was performed to assess cell proliferation following after knockdown of SAR1B as previously described. Briefly, lentivirus-infected RKO cells were seeded in 96-well plates at 3,000 cells/well. At 1, 2, 3, 4 and 5 days following incubation, 20 µl MTT solution (1 mg/ml) was added to each well and incubated at 37°C for 4 h. Subsequently, the medium was carefully removed and 100 µl acidic isopropanol (10% SDS, 5% isopropanol and 0.01 mol/l HCl) was added to each well. The plates were read using an automated microplate reader (Molecular Devices, LLC) at 490 nm.

Briefly, shSAR1B- or shCtrl-infected RKO cells were digested with trypsin and resuspended in standard medium after achieving the logarithmic growth phase. Cells were seeded into 96-well plates at a density of 2,000 cells/well. Fluorescence was detected every day using the Celigo system (Nexcelom Bioscience LLC) for 5 days continuously. Following adjustments in the parameters in analysis settings, the number of cells by scanning green fluorescence daily for 5 days at room temperature according to the manufacturer's instructions.

Apoptosis assessment was performed using an Annexin-V/FITC Apoptosis Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Briefly, cells were infected with shSAR1B or shCtrl. Cells were harvested and stained with Annexin-V/FITC and propidium iodide (PI) in binding buffer for 15 min at room temperature in the dark. The samples were analyzed using a BD FACScan system (BD Biosciences) to determine the percentage of cells exhibiting Annexin-V and PI staining. Apoptotic cells were subsequently analyzed via flow cytometry, using MoFlo XDP (Beckman Coulter, Inc.).

Cells were infected with shSAR1B or shCtrl. Protein was extracted using RIPA lysis buffer with proteinase inhibitor (Beyotime Institute of Biotechnology) and western blotting was performed as previously described (12). Protein concentrations of cell lysates were determined using the Bradford method. Equal amounts of protein (20 µg/lane) were separated using 12% SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked using 5% BSA, at 25°C for 2 h. After blocking at room temperature, the membrane was incubated with anti-SAR1B (1:1,000; cat. no. ab155278, Abcam) and anti-GAPDH (1:1,000; cat. no. sc-32233, Santa-Cruz Biotechnology, Inc.) primary antibodies at 4°C for 12 h. Membranes were washed three times with Tris-buffered saline Tween-20 buffer (TBST; 10 mM Tris, 150 mM NaCl, 0.05% Tween-20; Beijing Solarbio Science & Technology Co., Ltd.). Following the primary incubation, membranes were incubated with the horseradish peroxidase-conjugated (HRP) anti-mouse secondary antibody (1:5,000; cat. no. sc-2005; Santa-Cruz, Inc.) and anti-rabbit secondary antibody (1:5,000; cat. no. sc-2004; Santa-Cruz Biotechnology, Inc.) at 25°C for 2 h. Membranes were re-washed three times with TBST buffer. Electrochemiluminescence (cat. no. 6883; Cell Signaling Technology, Inc.) was used for visualization of the protein bands. GAPDH was used as the loading control and protein expression was quantified using ImageJ Software version 1.47 (National Institutes of Health).

The stable RKO cells after shRNA infection were used for the microarray analysis. Whole-genome gene expression levels of stable RKO cells following SAR1B knockdown were examined using the gene chip PrimeView Human Gene Expression Array (Thermo Fisher Scientific, Inc.). The sample labeling, microarray hybridization, washing and gene normalization were performed according to the manufacturer's protocols. Differentially expressed genes were subsequently identified via fold-change analysis and P-values; the threshold set for upregulated and downregulated genes was fold-change ≥1.5 and P<0.05. Cluster 3.0 software was used to analyze the differentially expressed genes, using hierarchical clustering method, and gene expression correlation coefficient as the distance, average connection. Then the Treeview software was used to draw the DEGs cluster map.

The present study used the Ingenuity Pathway Analysis system (https://www.nihlibrary.nih.gov/resources/tools/ingenuity-pathways-analysis-ipa) to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (21) and Gene Ontology (GO) (21,22) analyses of potential targets of SAR1B.

All experiments were performed in triplicate and were repeated at least three times. Data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student's t-test. The probability of survival was estimated using the Kaplan-Meier method. The log-rank test was used to statistically compare the differences in survival times. P<0.05 was considered to indicate a statistically significant difference.

The expression of SAR1B in CRC was analyzed using GEPIA datasets. As demonstrated in Fig. 1A and B, SAR1B expression was significantly upregulated in colon and rectum cancer samples compared with normal tissues.

Kaplan-Meier method was used to analyze the association between SAR1B expression levels and overall survival (OS) time in patients with CRC using two public datasets, GSE17536 and GSE17537. The results revealed that high expression of SAR1B was significantly associated with shorter OS time in patients with CRC (Fig. 1C and D).

In order to further validate the above analysis, 15 pairs of CRC samples were collected. As shown in Fig. 1E, RT-qPCR confirmed that SAR1B was significantly upregulated in CRC samples compared with in normal tissues (Fig. 1E).

RT-qPCR was used to determine the mRNA expression levels of SAR1B in CRC cell lines HCT116, SW620 and RKO. The results demonstrated that SAR1B was expressed in the three cell lines. The highest SAR1B mRNA expression was observed in SW620 cells, which was 7.56-fold higher than GAPDH expression (Fig. 2A). SAR1B mRNA expression levels were also high in HCT116 cells (7.35-fold higher compared with GAPDH). In addition, SAR1B mRNA expression was 7.23-fold higher than the level of GADPH in RKO cells, which was consistent with HCT116 and SW620 cells. The present study selected RKO cells for the validation of functional importance of SAR1B in CRC, which has been widely used in previous CRC studies (23–26).

To further explore the function of SAR1B in CRC, SAR1B was silenced in CRC RKO cells using lentivirus-mediated gene knockdown. As demonstrated in Fig. 2B, the strong and positive EGFP signals in RKO cells following infection with a lentivirus recombined with shSAR1B or shCtrl indicated that the efficiency of infection in RKO cells was high and thus qualified for subsequent experiments.

RT-qPCR and western blot analysis results revealed that knockdown of SAR1B was efficient at the mRNA and protein levels in shSAR1B-infected RKO cells (Fig. 2C and D). The RT-qPCR results demonstrated that the reduction efficiency reached 97% following infection with shSAR1B, which was significantly different compared with the shCtrl group (P<0.05; Fig. 2C). The western blotting results as also showed SAR1B protein was reduced in shSAR1B-infected cells (Fig. 2D). These results indicated that recombinant lentivirus targeting SAR1B effectively suppressed mRNA and protein expression of endogenous SAR1B in CRC cells.

In order to explore the potential functions of SAR1B in CRC, the DepMap database was used. The effects of SAR1B on cancer cell survival was analyzed using the DepMap portal. The results of genome-wide shRNA (or small interfering RNA) inhibition of SAR1B from the Broad (Fig. S1A), and Novartis (Fig. S1B) datasets showed the dependency of SAR1B for cancer cell survival, thus suggesting that SAR1B may promote the proliferation of CRC cells.

A Celigo cell counting application, which allows for direct imaging and counting of cells, was used to detect RKO cell proliferation following SAR1B knockdown. The data were collected for 5 days continually, and the results are presented in Fig. 3A. The results demonstrated that compared with cells infected with shCtrl, the green signals in cells infected with shSAR1B were significantly inhibited (P<0.05). These results suggested that the downregulation of SAR1B may inhibit cell proliferation (Fig. 3A). The average cell number was 12.76-fold higher on day 5 compared with that on day 1 in cells infected with shCtrl, whereas the average cell number was 4.74-fold higher on day 5 compared with that on day 1 in cells infected with shSAR1B, which was significantly different (P<0.001).

MTT and colony formation assays were used to further investigate the potential effects of SAR1B on the proliferation of RKO cells. A total of 2,000 cells were seeded and the cell numbers were analyzed using an MTT assay daily for 5 days (Fig. 3B). The proliferation was reduced in cells infected with shSAR1B compared with that in cells infected with shCtrl (P<0.05), which suggested that the rate of cell proliferation was significantly inhibited.

The colony formation assay also exhibited consistent inhibition of cell proliferation in shSAR1B-infected RKO cells. As demonstrated in Fig. 3C, the colonies were relatively smaller and lower in number in the shSAR1B group compared with the shCtrl group in RKO cells. Statistical analysis further revealed that knockdown of SAR1B significantly reduced the number of colonies formed by RKO cells (P<0.001; Fig. 3D). These results suggested that knockdown of SAR1B inhibited RKO cell proliferation.

To explore the role of SAR1B in apoptosis, flow cytometry was performed to determine the apoptotic rates in RKO cells infected with shCtrl or shSAR1B. As demonstrated in Fig. 4A and B, apoptosis was induced following shCtrl or shSAR1B infection. Further analysis revealed that the apoptotic rate was significantly reduced in the shCtrl group compared with the shSAR1B group, which was significantly different (P<0.01). These results suggested that SAR1B knockdown induced apoptosis of RKO cells, indicating the tumorigenic potential of SAR1B.

As the mechanisms underlying the effects of SAR1B on CRC progression remain largely unknown, microarray analysis was conducted. As presented in Fig. 5A, a total of 333 genes were upregulated and 369 genes were downregulated following SAR1B knockdown in RKO cells (Table SI).

KEGG pathway analysis revealed that SAR1B was significantly enriched in ‘TGF-β signaling’, ‘antiproliferative role of TOB in T cell signaling’, ‘paxillin signaling’, ‘glutamate degradation III (via 4-aminobutyrate)’, ‘molecular mechanisms of cancer’, ‘UDP-N-acetyl-D-glucosamine biosynthesis II’, ‘glutamine degradation I’, ‘HIPPO signaling’, ‘renal cell carcinoma signaling’, ‘RAR activation’, ‘epithelial adherens junction signaling’, ‘S-adenosyl-L-methionine biosynthesis’, ‘4-aminobutyrate degradation I’, ‘germ cell-sertoli cell junction signaling’, ‘BMP signaling pathway’, ‘hepatic fibrosis/hepatic stellate cell activation’, ‘FAK signaling’, ‘cell cycle regulation by BTG family proteins’ and ‘IGF-1 signaling’ (Fig. 5B). Of note, ‘IGF-1 signaling’ was significantly activated; >9 regulators of this pathway were upregulated following SAR1B knockdown in CRC by using Ingenuity Pathway Analysis system (https://www.nihlibrary.nih.gov/resources/tools/ingenuity-pathways-analysis-ipa) (Fig. 5C).

A PPI network regulated by SAR1B was constructed using the STRING database. This network contained 35 nodes. Among them, HOOK1, HOOK3, RAB9A, TBC1D15, SLC44A1, LRRC8B, SLC41A1, SCAMP1, SLC7A2, ADIPOR1, PAQR3, NUS1 and RDH10 were downregulated following SAR1B knockdown in RKO cells. Conversely, Akt, ARRDC3, ASAH2, ASAH2B, ceramidase, DENND6A, EFNB2, Ephb, KRIT1, LETM1, LRRC8A, MAGI2, MRS2, NT5E, RAB7, SLC25A20, SLC38A10, TBC1D7, TMED4, TRAK1, TRAK2 and vascular endothelial growth factor receptor were upregulated following SAR1B knockdown in RKO cells (Fig. 6).