CRC cells were seeded in 6-well plates 24 h before treatment. When the cell growth reached 70% confluence, 200 nm EXOs were added directly to the cells, and the control group was added to PBS. After 48 h of treatment, cells were collected for further experiments.

Cell counting kit 8 (CCK-8) assay

CRC cells (2 × 103 cells/well), prepared as required, were plated in a 96-well plate. After incubating for 0, 1, 2, 3, and 4 days, 10 µL of CCK-8 solution (Beyotime Biotechnology, Shanghai, China) was added and incubated for 3 h. Absorbance was subsequently measured at 450 nm using a spectrophotometer (Multiscan MK3, Thermo Fisher Scientific, Waltham, MA, USA).

Transwell assay

Transwell chambers (8 μm; Corning, NY, USA) were used to assess migration and invasion capability of CRC cells. CRC Cells (3 × 104 cells/well) were added to a serum-free medium (Gibco). The lower chamber contained 600 µL of 12% FBS (Gibco). After 24 h of incubation, the migrated cells were fixed in 4% formaldehyde (Meilunbio, Dalian, China) for 30 min and stained with 0.2% crystal violet (Solarbio). For the invasion experiments, Matrigel (Corning) was pre-diluted and applied to the upper chamber before conducting the invasion assays. Photomicrographs were captured using a microscope (Olympus). The number of cells was quantified using Image J software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA).

Colony-formation assay

Cells (200 cells/well) were seeded in a 6-well plate, and the plates were incubated for two weeks at 37 °C. The culture medium was changed every 3 days. The cells were constantly monitored until colonies developed. The cells were then stained with 0.1% crystal violet (Solarbio, Beijing, China) for 30 min. Photomicrographs were captured using a microscope (Olympus). The number of cells was quantified using ImageJ software (version 1.8.0, National Institutes of Health). A clone was defined as one containing ≥ 50 cells.

Sphere-formation assay

CRC cells (1 × 103 cells/well) were grown in serum-free RPMI 1640 medium (Gibco) in ultra-low-adhesion plates (Corning). The medium was changed every 2 days. Two weeks later, spheres were counted using a microscope (Olympus), and images were acquired.

Dual-luciferase assay

The psiCHECK dual-luciferase reporter system (Thermo Fisher Scientific) was used in this study. Wild-type (WT) and mutant NEK2 were amplified and inserted into the psiCHECK2 vector. Subsequently, a 24-well plate was seeded with 4 × 104 HEK293T cells, which were allowed to adhere overnight. Plasmids containing the Renilla luciferase expression plasmid were transfected using Lipofectamine 2000. Luciferase activity ratios were assessed using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA).

RNA immunoprecipitation (RIP) assay

To understand the relationship between miR-486-5p and NEK2, we performed RIP experiments. Magnetic bead-antibody complexes were first prepared using protein A/G agarose beads (Thermo Fisher Scientific) and NEK2 antibody (Abcam, Cambridge, MA, USA). The cells were lysed and immunoprecipitated with the complexes. Purified RNA was examined by qRT-PCR after washing the unbound material with PBS.

Quantitative real-time-PCR (qRT-PCR)

TRIzol reagent was used to extract total RNAs from designated cells. After RNA extraction, cDNA was synthesized using the SuperScript II First-Strand cDNA Synthesis Kit (Invitrogen Life Technologies). Following cDNA synthesis, PCR amplification was carried out on an ABI 7500 Real-Time PCR System (Applied Biosystems) using the SYBR Green Master Mix obtained from Applied Biosystems (Foster City, CA). The relative expression levels of target genes were determined using the comparative 2−ΔΔCT method. β-actin served as a loading control. The primers sequences are followed as: miR-486-5p forward primer 5’-GCCGTCCTGTCATGAGCTGC-3’, reverse primer, 5’-GTGCAGGGTCCGAGGT-3’; miR-4448 forward primer 5’-GCGACGAGGCTCCTTGGT-3’, reverse primer, 5’-TATGGTTGTTCACGACTCCTTCAC-3’; U6 forward primer, 5’-GCTTCGGCAGCACATATACTAAAA-3’, reverse primer, 5’- GCTTCGGCAGCACATATACTAAAAT-3’; NEK2 forward primer, 5’-CCTGGAGCAGAAGGAACGTG-3’, reverse primer, 5’-TGGCTGAGGATGGAAGATCAAG-3’; β-actin, forward primer, 5’- CTCCATCCTGGCCTCGCTGT-3’, reverse primer, 5’- GCTGTCACCTTCACCGTTCC-3’.

Western blot

Proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) on ice. Next, 20 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following the electrophoresis, the isolated proteins were carefully transferred onto a PVDF membrane (0.45 μm; Millipore). Membranes were blocked with 5% milk for 2 h. After the blocking step, the membrane was incubated overnight with primary antibodies at 4 °C, followed by a 1 h incubation with secondary antibodies at 22 °C. The ECL program (Beo Tianmei Biotechnology, Shanghai, China) was used to observe the results. The antibodies used were as follows: anti-CD63 (1:1000 dilution, ab134045, Abcam), anti-CD81 (2 µg/ml, ab79559, Abcam), anti-TSG101(1:1000 dilution, ab125011, Abcam), anti-Nanog (1:5000 dilution, ab109250, Abcam), anti-OCT4 (1:1000 dilution, ab181557, Abcam), anti-NEK2 (1:1000 dilution, ab227958, Abcam), IgG (ab172730, Abcam), and anti-β-actin (1 µg/ml, ab8226, Abcam).

The indicators of glucose metabolism

To investigate cellular glucose metabolism, we utilized the Glucose Uptake Colorimetric Assay Kit (BioVision, Milpitas, CA) and Lactate Assay Kit II (BioVision, Palo Alto, California, USA) to measure both glucose uptake and lactate production levels. A spectrophotometer (Multiscan MK3, Thermo Fisher Scientific) was used to measure the absorbance of the samples. Subsequently, we calculated glucose uptake and lactate production levels by comparing the absorbance values to a standard curve.

Extracellular acidification rate (ECAR)

ECAR was performed using a Seahorse Extracellular Flux Analyzer XF96 (Seahorse Bioscience, North Billerica, MA, USA). Overnight-incubated 96-well plates, seeded at a density of 2 × 104 cells per well, were subjected to sequential introduction of glucose, oligomycin (an inhibitor of oxidative phosphorylation), and 2-DG (a glycolysis inhibitor) to assess ECAR (mpH/min). Data analysis was performed using Seahorse XF-96 software.

Statistical analysis

GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA) and SPSS 26.0 (IBM Corporation, Chicago, Illinois, USA) were used for data analysis. All experiments were repeated three times. The results were presented in terms of mean ± standard deviation (SD). For two group comparisons, the unpaired Student’s t-test was employed, while ANOVA was utilized for multiple groups. An indication of statistical significance was a P-value < 0.05.