The HCT116, HT29, RKO and SW480 human colorectal cancer cell lines were purchased from Procell Biotechnology Co,. Ltd. Authentication of the aforementioned cell lines was conducted using short tandem repeat profiling. HUVECs were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, CAMS/PUMC. HCT116 and RKO cell lines were cultured with Dulbecco's modified Eagle's medium (Sangon Biotech Co., Ltd.) containing 10% fetal bovine serum (FBS) (PAN-Biotech GmbH) and 1% penicillin/streptomycin (Sangon Biotech Co., Ltd.). HT29 and SW480 cell lines were cultured in RPMI-1640 (Sangon Biotech Co., Ltd.) containing 10% FBS and 1% penicillin/streptomycin. HUVECs were cultured in a HUVEC specific complete medium (Procell Life Science & Technology Co., Ltd.). All cells were maintained in a humidified atmosphere at 37°C containing 5% CO₂.

A total of three different siRNAs (50 µM) of 5-LOX were synthesized to knock down the expression of 5-LOX in HCT116 cells, and a non-targeting siRNA control (si-NC) was transfected to the cells as a negative control. These sequences were synthesized by Huzhou Hippo Biotechnology Co., Ltd. as follows: si-5-LOX-#1, 5′-CCAAAUGCCACAAGGAUUUTT-3′ (sense) and 5′-AAAUCCUUGUGGCAUUUGGCA-3′ (antisense); si-5-LOX-#2, 5′-GCAGGAAGACCUGAUGUUUTT-3′ (sense); and 5′-AAACAUCAGGUCUUCCUGCCA- 3′ (antisense); si-5-LOX-#3, 5′-CCAUUGCAAUCAACACCAATT-3′ (sense); and 5′-UUGGUGUUGAUUGCAAUGGTG-3′ (antisense); si-NC, 5′-UUCUCCGAACGUGUCACGUdTdT-3′ (sense) and 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (antisense). HCT116 cells in the logarithmic growth phase were seeded into six-well plates at a density of 2×10⁵ per well. Transfection with 40 nM siRNA was performed with Lipofectamine RNAiMAX transfection reagent (cat. no. 13778030; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. Following transfection at 37°C for 48 h, gene knockdown efficiency was measured by quantitative PCR (qPCR) and western blotting analysis.

Recombinant lentiviruses overexpressing 5-LOX (OE-5-LOX) were constructed by using the GV365 plasmid (pUbi-MCS-3FLAG-pCMV-EGFP), with empty vector control lentiviruses (OE-Vector) as control. All lentiviruses were produced by Shanghai GeneChem Co., Ltd.. The lentiviruses were generated by transfecting GV365, pHelper 1.0 and pHelper 2.0 plasmid (Shanghai GeneChem Co., Ltd.) into 293T cells with E-trans transfection reagents (cat. no. REVG007; Shanghai GeneChem Co., Ltd.) at 37°C for 48 h. The titer of the lentivirus used for infection was 2×10⁸ transducing units/ml. RKO cells in the logarithmic growth phase were seeded into six-well plates at a density of 2×10⁵/well. Lentiviruses were infected into the RKO cells at a multiplicity of infection of 50, supplemented with 5 µg/ml polybrene (MilliporeSigma) to enhance infection efficiency. After incubation at 37°C for 72 h, infection efficiency was assessed by fluorescence microscopy. Successful overexpression of the target 5-LOX gene was verified by qPCR and western blotting analysis.

HCT116 cells (1×10⁶ cells/well) were transfected with siRNA and RKO cells (1×10⁶ cells/well) were infected with lentivirus (as aforementioned). Following siRNA transfection (48 h) and lentivirus infection (72 h), the culture media was replaced with fresh media; conditioned media were subsequently collected after 24 h of incubation. HUVECs (1×10⁶ cells/well) were plated in 6-well plates and incubated with the media collected from siRNA-transfected HCT116 or lentivirus-infected RKO cells at 37°C for 24 h. HUVECs were then divided into four groups: si-NC group (transfected with the non-targeting siRNA control) and si-5-LOX-#2 group (transfected with si-5-LOX-#2), OE-Vector group (infected with empty vector control lentiviruses) and OE-5-LOX group (infected with 5-LOX overexpressing lentiviruses). Consequently, qPCR was used to detect the mRNA levels of VEGF and angiogenin in HUVECs. The expression of VEGF protein in HUVECs was determined by western blotting analysis.

Cell viability was investigated using the cell counting kit (CCK)-8 assay (cat. no. C0037; Beyotime Biotechnology). HUVECs (2,500 cells/well) were cultured with media collected from siRNA-transfected HCT116 cells or lentivirus-infected RKO cells in a 96well culture plate at 37°C for 24, 48 and 72 h. The absorbance was detected at 450 nm after the cells had been treated with 10% CCK-8 at 37°C for 2 h. Cell viability was calculated as the ratio of optical density (OD) values of medium-treated cells (24, 48 and 72 h) to those of naive cells (0 h).

HCT116 cells transfected with siRNA were divided into si-NC and si-5-LOX#2 groups. RKO cells infected with lentiviruses were divided into OE-Vector group and OE-5-LOX group. HCT116 cells (8×10⁴ cells/well) and RKO cells (4×10⁴ cells/well) were suspended in serum-free medium and deposited into the upper Transwell chamber. Upper chambers without Matrigel or Matrigel-coated were used for migration and invasion assays, respectively. For the invasion assay, Matrigel (Corning, Inc.) was diluted in serum-free medium at a ratio of 1:8. A uniform layer was applied to the upper chamber of the Transwell inserts, and the coated inserts were incubated at 37°C for 1 h to allow polymerization. A medium containing 10% FBS was added to the lower chamber for the two assays. Cells were cultured at 37°C and 5% CO₂ for 24 h and then cleaned with PBS. After discarding the PBS, cells were fixed with methanol (MilliporeSigma) for 15 min at room temperature and then stained with 0.1% crystal violet (MilliporeSigma) for 15 min at room temperature. Stained cells were imaged using a light microscope (×100 magnification), and a total of five random fields per insert were captured.

Transfected HCT116 cells and infected RKO cells were seeded at a density of 1×10⁶ cells/well in a six-well culture plate attaining 90% confluence, then a wound was created using a 200 µl pipette tip. Images were captured at three random fields of view by a light microscope (×40 magnification) at 0 and 24 h. Wound area was measured using ImageJ software (version 1.54g; National Institutes of Health). Wound-healing assay results were presented as migration rate (%)=(initial wound area-wound area at 24 h)/initial wound area ×100.

Transfected HCT116 cells and infected RKO cells were collected and suspended in Binding Buffer at a density of 1×10⁶ cells/ml. HCT116 and RKO cells were stained with Annexin V-FITC and Annexin V-APC Apoptosis Detection kits (cat. no. 88-8005-74/88-8007-74; Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. Apoptosis was examined by flow cytometry (cytoFLEX; Beckman Coulter, Inc.). The apoptotic rate was assessed with CytExpert (V 2.4.0.28) software (Beckman Coulter, Life science).

Total RNA of HCT116, HT29, RKO, SW480, transfected HCT116, infected RKO and HUVECs was extracted by using RNAprep Pure Cell/Bacteria Kit (cat. no. DP430; Tiangen Biotech Co., Ltd.) in accordance with the manufacturer's instructions. RT was performed with a ReverTra Ace qPCR RT kit (cat. no. FSQ-101; Toyobo Co., Ltd.) in a total volume of 20 µl. RT was performed as follows: 37°C for 15 min and 98°C for 5 min. The qPCR was performed with SYBR Green qPCR Master Mix (cat. no. HY-K0522; MedChemExpress). The reaction protocol was as follows: 5 min initial denaturation at 95°C, followed by 40 cycles of amplification with 15 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C for each cycle. This was followed by a melt curve analysis to determine the reaction specificity. β-actin was used as the endogenous control. The oligonucleotide primers used were as follows: β-actin, 5′-CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse); 5-LOX, 5′-ACAAGCCCTTCTACAACGACT-3′ (forward) and 5′-AGCTGGATCTCGCCCAGTT-3′ (reverse); Angiogenin, 5′-GTTGGTCTTCGTGCTGGGTCTG-3′ (forward) and 5′-AGTGCTGGGTCAGGAAGTGTGT-3′ (reverse); VEGF, 5′-GGCAGAAGGAGGAGGGCAGAAT-3′ (forward) and 5′-GGGCACACAGGATGGCTTGAAG-3′ (reverse). The 2^−ΔΔCq method was used to calculate the relative RNA expression (24).

HCT116, HT29, RKO, SW480, transfected HCT116 and infected RKO cells were incubated and then centrifuged at 175 × g to collect cells. Total protein was extracted using RIPA buffer (Beyotime Biotechnology). Protein concentration was determined with a BCA protein quantification kit (cat. no. P0009; Beyotime Biotechnology) in accordance with the manufacturer's instructions. Extracted proteins (20 µg) were then separated by SDS-PAGE in 10–12% acrylamide gel and transferred to a polyvinylidene fluoride membrane (MilliporeSigma). The membrane was subsequently blocked with 5% skimmed milk in TBS containing 0.05% Tween-20 (Beyotime Biotechnology) for 1 h at room temperature. The membrane was then incubated with a primary antibody (1:1,000) overnight at 4°C. The primary antibodies were 5-LOX (cat. no. 83794-4-RR; Proteintech Group, Inc.), E-cadherin (cat. no. 20874-1-AP; Proteintech Group, inc.), N-cadherin (cat. no. 13116; Cell Signaling Technology, Inc.), Snail (cat. no. 13099-1-AP; Proteintech Group, Inc.), Vimentin (cat. no. 5741; Cell Signaling Technology, Inc.), VEGFA (cat. no. 81323-2-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 81640-5-RR; Proteintech Group, Inc.). The membrane was washed three times in TBST and incubated with diluted secondary antibody (1:10,000) conjugated with horseradish peroxidase solution (cat. no. ab205718; Abcam) for 1 h at room temperature. After washing, the membrane was visualized with ECL substrate (cat. no. SW2050; Beijing Solarbio Science & Technology Co., Ltd.) by using chemiluminescence (AzureC500; Azure Biosystems, Inc.). The gray values of the bands were quantified using ImageJ (version 1.54g; National Institutes of Health), and GAPDH was used as the loading control.

RKO cells infected with lentiviruses (the OE-Vector and OE-5-LOX groups) were seeded in six-well plates at a density of 5×10⁵ cells/well. After 72 h of incubation at 37°C, media were replaced with fresh media, and the cells were cultured at 37°C for another 48 h before supernatant collection. The concentrations of leukotriene E₄ (LTE₄) and leukotriene B₄ (LTB₄) in the supernatant were detected using a LTE₄ ELISA kit (cat. no. MM-928804O1) and a LTB₄ ELISA kit (cat. no. MM-927697O1; both Jiangsu Enzyme Immunoassay Co., Ltd.), in accordance with the manufacturer's instructions. OD values were measured at 450 nm, and sample concentrations were calculated based on the standard curve. Relative expression was calculated as concentration ration of the OE-5-LOX group relative to the OE-Vector group.

Data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism statistical package (version 9.5; Dotmatics). Comparisons between two groups were conducted with Student's t-test. Analysis of more than two groups was performed with one-way ANOVA followed by Dunnett's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

To select the prime candidate CRC cell line for further 5-LOX gene knockdown and overexpression, the mRNA and protein expression levels of 5-LOX were analyzed in four CRC cell lines: HCT116, HT29, RKO and SW480. As shown in Fig. 1A, high levels of 5-LOX mRNA were observed in HCT116 and HT29 cells while low levels of 5-LOX mRNA were observed in RKO and SW480 cells. The 5-LOX protein was widely expressed in HCT116, HT29 and SW480 cells other than RKO cells (Fig. 1B). Thus, HCT116 cells, which exhibited high levels of 5-LOX expression, were used for the subsequent gene knockdown investigations. RKO cells, with low levels of 5-LOX expression, were used for gene overexpression investigations.

The mRNA levels of 5-LOX in HCT116 cells were determined by RT-qPCR following treatments with siRNA for 48h. As shown in Fig. 2A, three different siRNAs inhibited the 5-LOX mRNA levels up to 72% compared with the si-NC group. Of these three siRNAs, si-5-LOX-#2 inhibited the expression of 5-LOX protein more effectively than the other siRNAs (Fig. 2B). Therefore, the following investigations were conducted by using si-5-LOX-#2.

Recombinant lentiviruses expressing 5-LOX were used to infect RKO cells. An embedded green fluorescent protein-tag was used to visualize viral infection. As shown in Fig. 3A, RKO cells were successfully infected with the recombinant lentiviruses after 72 h of incubation. The mRNA level of 5-LOX in the OE-5-LOX group was significantly higher than that in the OE-Vector group (Fig. 3B). As shown in Fig. 3C, the expression of 5-LOX protein in the OE-Vector group remains at the basal level, consistent with the inherently low endogenous 5-LOX expression in RKO cells. However, the expression of 5-LOX protein in the OE-5-LOX group was significantly higher than that in OE-Vector group. 5-LOX is a key enzyme in leukotriene biosynthesis (25); as shown in Fig. S1, the overexpression of 5-LOX significantly increased levels of LTB₄ and LTE₄ in RKO cells.

Transwell migration (Fig. 4A and B) and wound healing assays (Fig. 4I) revealed that the migration ability of HCT116 cells in the si-5-LOX-#2 group was significantly reduced compared with the si-NC group. Transwell invasion assay revealed that the si-5-LOX-#2 group had a significantly lower number of invaded cells than the si-NC group (Fig. 4C and D). However, the migration ability of RKO cells in the OE-5-LOX group was significantly higher than that in the OE-Vector group which was determined by Transwell migration (Fig. 4E and F) and wound healing assays (Fig. 4J). RKO cells overexpressing 5-LOX (the OE-5-LOX group) exhibited significantly higher invasion (Fig. 4G and H) ability compared with the cells infected with the control-lentiviruses (the OE-Vector group).

As shown in Fig. 5A, the apoptotic rate of 5-LOX-silenced HCT116 cells (the si-5-LOX-#2 group) was significantly increased compared with the cells transfected with control siRNA (the si-NC group). Alternatively, RKO cells overexpressing 5-LOX (the OE-5-LOX group) exhibited significantly lower apoptotic rate in comparison to the OE-Vector group (Fig. 5B).

To evaluate the relationship between 5-LOX and EMT in CRC cells, the expression of molecular markers for EMT were analyzed by WB in 5-LOX-silenced CRC cells, 5-LOX-overexpressed CRC cells and their corresponding control cells. WB analyses revealed that 5-LOX knockdown significantly increased the expression of E-cadherin and reduced the expression of N-cadherin, Snail and Vimentin in HCT116 cells (Fig. 6A). However, the overexpression of 5-LOX significantly downregulated the expression of E-cadherin but upregulated the expression of N-cadherin, Snail and Vimentin in RKO cells (Fig. 6B).

To elucidate whether the products secreted by 5-LOX-silenced and 5-LOX-overexpressed CRC cells affect the angiogenesis of HUVECs, HUVECs were treated with media from 5-LOX-silenced HCT116 cells, 5-LOX-overexpressed RKO cells and their corresponding control cells in vitro. The CCK-8 assay showed that cell viability of HUVECs was significantly suppressed by treating with culture medium from the si-5-LOX-#2 group (Fig. 7A), while cell viability of HUVECs was significantly promoted by treating with culture medium from the OE-5-LOX group (Fig. 7B). The mRNA levels of VEGF and angiogenin were analyzed by RT-qPCR; as shown in Fig. 7C, the mRNA levels of VEGF and angiogenin were significantly lower in HUVECs which had been treated with culture medium from the si-5-LOX-#2 group than in HUVECs which had been treated with culture medium from the si-NC group. However, the mRNA levels of VEGF and angiogenin were significantly increased when HUVECs were treated with culture medium from the OE-5-LOX group rather than with culture medium from the OE-Vector group (Fig. 7D). The expression of VEGF protein was significantly decreased in HUVECs treated with conditioned media from 5-LOX silenced HCT116 cells but significantly increased in HUVECs treated with conditioned media from 5-LOX overexpressed RKO cells, compared with their respective control groups (Fig. S2).