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. 2024 Nov 6;24:1356. doi: 10.1186/s12885-024-13086-9

Mesenchymal stem cell-derived exosomes carrying miR-486-5p inhibit glycolysis and cell stemness in colorectal cancer by targeting NEK2

Facai Cui 1,✉, Yu Chen 2, Xiaoyu Wu 1, Weifeng Zhao 3
PMCID: PMC11539302  PMID: 39506654

Abstract

Colorectal cancer (CRC) is a major global concern. Mesenchymal stem cell-derived exosomes (MSC-EXOs) have demonstrated efficacy as a therapeutic approach for colorectal cancer. However, the precise mechanism by which MSC-EXOs treat colorectal cancer remains unclear. Human umbilical cord (hUC)-MSC-EXOs were isolated and identified. Cell Counting Kit-8 (CCK-8), Transwell, and colony formation assays were used to assess the activity of CRC cells. Glucose consumption, lactic acid production, and extracellular acidification rate (ECAR) were measured to assess glycolytic activity. Cell stemness was assessed using a sphere-formation assay. Furthermore, MSC-exosomal microRNAs (miRNAs) in CRC tissues were analyzed using the EVmiRNA database, and aberrantly expressed miRNAs in CRC cells were obtained from the Gene Expression Omnibus (GEO) database. The binding relationship between miR-486-5p and the never in mitosis gene A-related kinase 2 (NEK2) was predicted using the Starbase database and validated through RNA binding protein immunoprecipitation (RIP) and dual luciferase assays. These results showed that hUC-MSC-EXOs inhibited the proliferation and metastasis of CRC cells. Moreover, glycolysis and stemness abilities of CRC cells also decreased after treatment with hUC-MSC-EXOs. miR-486-5p was found to be enriched in hUC-MSC-EXOs and significantly downregulated in CRC cells. miR-486-5p directly bound to NEK2. Overexpression of NEK2 reversed the inhibitory effect of miR-486-5p on CRC cell glycolysis and stemness. Our study highlights that hUC-MSC-EXO miR-486-5p inhibits glycolysis and cell stemness in CRC by targeting NEK2. This finding offers compelling evidence supporting the potential application of hUC-MSC-EXOs in the treatment of CRC.

Keywords: Colorectal cancer, hUC-MSC-EXOs, miR-486-5p, NEK2, Glycolysis, Stemness

Introduction

Colorectal cancer (CRC) is among the most frequently diagnosed cancers worldwide [1]. CRC is divided into adenocarcinoma, mucinous adenocarcinoma, and undifferentiated carcinoma. Adenocarcinoma is common and accounts for 90% of all CRC cases. Research has shown that CRC was related to age, diet, obesity, and genetics. Accumulation of genetic mutations has been shown to facilitate malignant lesions [2]. Currently, the management of patients with unresectable or metastatic disease is surgery for patients diagnosed with limited metastatic and systemic (such as chemotherapy, immunotherapy, and targeted agents) disease [3]. Despite a 90% 5-year survival rate for CRC patients [4], the incidence of CRC has been increasing, with a projected 27.7% increase among individuals aged 35–49 years by 2030 [4]. As such, the identification of molecular biomarkers and exploration of innovative treatment approaches for CRC are essential for reducing the health burden on individuals.

In recent years, mesenchymal stem cells (MSCs), which are widely distributed throughout the body, have been used in the diagnosis and treatment of tumors owing to their unique advantages [5]. MSCs have been found to carry exosomes (EXOs) and other extracellular vesicles (EVs) to participate in the regulation of malignant progression of tumors [6]. EXOs are 30–100 nm EVs that contain a high concentration of proteins, lipids, nucleic acids, and other constituents. They can act as messengers for the transfer of intercellular signals, thereby affecting the behavior and functionality of recipient cells [7]. Compared to the direct use of stem cells, using EXOs for therapy offers the advantage of avoiding concerns related to cell heterogeneity and the risk of tumor formation [8]. Intriguingly, MSC-EXOs can carry microRNAs (miRNAs) that are involved in regulating multiple tumor functions [9]. Bone marrow (BM)-MSC-EXOs displayed inhibitory efficacy against hepatocellular carcinoma by releasing miR-205-5p [10]. Human umbilical cord (hUC)-MSC-EXOs miR-451a inhibited hepatoma growth and chemotherapy resistance [11]. However, the specific mechanisms of the components of EXO-miRNAs are still not fully understood.

In view of previous reports, this study aimed to demonstrate the role of human umbilical cord (hUC)-MSC-EXOs in regulating glycolysis and the stemness of CRC cells. Our study identified miR-486-5p as an MSC-EXO miRNA that was poorly expressed in CRC cells. In previous studies miR-486-5p, it was shown to be poorly expressed in CRC tissues [12]. miR-486-5p was also regarded as a tumor suppressor that inhibits the proliferation and migration of CRC cells [13]. However, its regulatory effects on other functions of CRC cells have not been explored. In addition, the never in mitosis gene A-related kinase 2 (NEK2) was shown to be a target for miR-486-5p in this study. NEK2 has been shown to promote glycolysis of cancer cells in several malignancies [14, 15]. NEK2 has also been found to play an important role in inducing cancer cells [16]. These outcomes imply that MSC-EXO miR-486-5p may regulate glycolysis and stemness of CRC cells by mediating the transcription of NEK2. Bioinformatics analysis combined with in vitro experiments further verified this hypothesis. This study provides potential therapeutic targets for CRC from the perspective of stem cell therapies.

Materials and methods

Bioinformatics analysis

miRNAs in MSC-EXOs and CRC tissues were searched using the EVmiRNA database (http://bioinfo.life.hust.edu.cn/EVmiRNA/#!/). Differentially expressed (DE) miRNAs between CRC and normal colon cells were analyzed using the GSE40247 dataset from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database. Meanwhile, the starBase, miRDB, and mirDIP databases were employed to predict the target genes of miR-486-5p, which were then intersected with upregulated DEmRNAs in CRC cells identified from the GSE202408 dataset.

Cell culture

The human colorectal cancer cell lines SW480 and LOVO, HEK293T cells, the normal colon cell line FHC, and hUC-MSCs were acquired from the American Type Culture Collection (ATCC). hUC-MSCs were cultured in a serum-free medium (Gibco, Grand Island, NY, USA). Roswell Park Memorial Institute (RPMI)-1640 (Gibco) medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin was used to culture CRC and normal colon cell lines. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin solution. The culture conditions for these cells were 37 °C and 5% CO2.

Cells transfection

The miR-486-5p inhibitor, miR-486-5p mimic, negative control (NC mimic and NC inhibitor), and NEK2 overexpressing plasmid vector were sourced from GenePharma (Shanghai, China). CRC cells were seeded in 6-well plates overnight (1 × 106 cells/well). CRC cells were transfected with 20 nM miR-486-5p mimic, NC mimic, miR-486-5p inhibitor, NC inhibitor and 2.0 µg NEK2 overexpressing vector using Lipofectamine 3000 (Invitrogen Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions.

The isolation of EXOs

The supernatant of cultured hUC-MSCs was subjected to ultracentrifugation to separate and extract EXOs. Briefly, after centrifuging the hUC-MSC suspension at 2000 × g for 15 min, debris and apoptotic bodies were removed by centrifugation at 10,000 × g for 30 min. To extract EXOs, the supernatant was centrifuged at 110,000 × g for 80 min. After washing with phosphate-buffered saline (PBS), the separated EXOs were centrifuged for 60 min at 110,000 × g. Finally, the EXOs underwent through a 0.22 μm filter (Millipore, Bedford, MA, USA) after being resuspended in PBS.

Transmission electron microscope (TEM)

The EXOs that were isolated underwent a process where they were placed on a formvar-coated copper grid and fixed using 2% paraformaldehyde for 10 min. The EXOs were embedded in a mixture of 0.13% methylcellulose and 0.4% uranyl acetate. Subsequently, electron microscopy (HITACHI, Tokyo, Japan) at 80 V was used to examine the grid, and the images were captured during the process.

Nanoparticle tracking analysis (NTA)

The EXO size distribution was ascertained using a Nanoparticle Tracking Analyzer (Zeta View, Particle Metrix, Dusseldorf, Germany). EXOs were diluted with 1× PBS to a suitable concentration, with the number of EXO samples between 50 and 400. The sizes of the EXO were examined according to the instrument.

PKH-26 labeled EXOs

The PKH-26 kit (Solarbio, Beijing, China) was used to label the EXOs and observe their uptake in CRC cells. First, 4 µL of PKH-26 ethanol solution was added to 1 ml of dilution C and mixed to form a staining solution. Exosomes were incubated with an equal volume of staining solution for 3 min. After immediately terminating the staining by adding 2 mL of FBS, the solution was centrifuged at 100,000 × g for 1 h. PKH-26 labeled EXOs were obtained by washing with PBS and resuspension in 5 mL of complete medium. Under a fluorescence microscope, the CRC cells were cultivated with PKH-26 labeled EXOs for 24 h, and the resulting images were viewed.

EXO treated cells

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.

Results

The characterization of hUC-MSC-EXOs

The EXOs derived from hUC-MSCs were certified. Upon examination using TEM, the isolated EXOs displayed a disc-shaped structure and were enveloped by double layer membranes (Fig. 1A). NTA results showed that most EXOs were between 50 and 150 nm in diameter (Fig. 1B). Furthermore, CD63, CD81, and TSG101, which were EXO-specific protein biomarkers, were identified within EXOs (Fig. 1C). After incubating the SW480 and LOVO cell lines with EXOs labeled with PKH-26, red fluorescence was detected in the cytoplasm of the CRC cells, as illustrated in Fig. 1D. This observation suggested that CRC cells successfully internalized EXOs.

Fig. 1.

Fig. 1

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The hUC-MSC-EXO characterization. (A) The images of EXOs extracted from hUC-MSC were detected by TEM. (B) The sizes of EXOs secreted by hUC-MSC were detected using NTA. (C) Western blot was applied to measure the hUC-MSC and hUC-MSC-EXO biomarkers. (D) Exosomes characterized with PKH-26 were detected

hUC-MSC-EXOs inhibits proliferation, migration, glycolysis and cell stemness in CRC cells

Upon co-cultured separately with SW480 and LOVO cells. After 48 h of co-culture, the CCK-8 assay was used to measure the proliferation capacity of the cells in the EXO and PBS groups. The results showed that the OD values of cells in the EXO group were significantly downregulated on the second day compared with those in the control group. With the increase in cell culture days, the proliferation activity of cells in the EXO group gradually decreased compared to that in the PBS group (Fig. 2A). Concurrently, a decrease in the colony formation capacity of CRC cells was also noted (Fig. 2B). Exosome treatment reduced the migratory and invasive capacity of CRC cells (Fig. 2C-D). Glucose consumption and lactate production, which are crucial indicators of glycolytic capacity, were assessed. The results revealed that EXOs inhibited glucose consumption and lactate production (Fig. 2E-F). The level of ECAR markedly decreased in CRC cells upon EXO treatment, indicating a decline in cellular glycolytic capacity (Fig. 2G). Additionally, alterations in the stemness of CRC cells were observed with decreased sphere-forming ability (Fig. 2H). The expression of Nanog and OCT4 proteins was significantly reduced when CRC cells were co-cultured with EXOs (Fig. 2I). This implies that EXOs may influence the stemness of CRC cells by affecting their glycolytic capacity of cancer cells.

Fig. 2.

Fig. 2

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hUC-MSC-EXOs inhibit proliferation, migration, glycolysis, and stemness in CRC cells. hUC-MSC-EXOs were employed to treat the CRC cells. (A) The CCK-8 assay was carried out to gauge the CRC cells’ ability to growth. (B) The CRC cells’ capacity for colony formation was evaluate. (C–D) The Transwell test was employed to identify the cells’ capacity for migration and invasion. (E-G) Glucose consumption, lactic acid production, and the level of ECAR were detected by kits. (H) The sphere formation ability was tested in CRC cells. (I) Western blot was applied to identify the CRC cells’ stemness marker proteins. * represents P < 0.05 compared with the PBS group

Mir-486-5p is highly expressed in hUC-MSC-EXOs and released into CRC cells

Having established that hUC-MSC-EXOs could influence the function of CRC cells, we sought to further investigate their potential molecular mechanisms. First, we obtained a total of 108 miRNAs in MSC-EXOs and 361 miRNAs in colon tissues according to the data available in the EVmiRNA database, of which 52 were intersecting (Fig. 3A). Next, data on a GEO microarray GSE40247 based were analyzed, and 53 miRNAs were found to be significantly downregulated in CRC cells. By intersecting 52 EVmiRNAs with 53 significantly downregulated miRNAs in CRC cells, two miRNAs (miR-4448 and miR-486-5p) were obtained (Fig. 3B). Furthermore, we measured the expression levels of miR-4448 and miR-486-5p in FHC and CRC cells, respectively. Compared with normal colon cancer cells (FHC), only miR-486-5p expression was significantly downregulated in CRC cells (Fig. 3C). MiR-486-5p was regarded as the target miRNA for subsequent studies. Remarkably, miR-486-5p was highly expressed in hUC-MSC-EXOs, indicating its potential significance as a target for EXO-mediated regulation of CRC cells (Fig. 3D). When CRC cells were treated with EXOs, the expression of miR-486-5p was significantly upregulated in both 2 types of CRC cells (Fig. 3E).

Fig. 3.

Fig. 3

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miR-486-5p is abundantly expressed in hUC-MSC-EXOs and released into CRC cells. (A) Intersected miRNAs found in MSC-EXOs and in colon tissues. (B) Venn diagram of 52 EVmiRNAs with 53 miRNAs down-regulated in CRC cells. (C) qRT-PCR was used to assess the expression of miR-486-5p and miR-4448 in FHC, SW480 and LOVO cells. (D) qRT-PCR was utilized to measure the level of miR-486-5p in hUC-MSC and hUC-MSC-EXOs. (E) qRT-PCR was used to assess the expression of miR-486-5p in CRC cells treated with hUC-MSC-EXOs. ns represents no difference compared with the FHC group; * represents P < 0.05 compared with the MSC, FHC and PBS groups

hUC-MSC-EXOs inhibits glycolysis and cell stemness of CRC cells via regulating the expression of miR-486-5p

An miR-486-5p inhibitor was introduced to confirm the role of miR-486-5p in CRC cells. As shown in Fig. 4A, the level of miR-486-5p was suppressed after treatment with the miR-486-5p inhibitor. The results of the CCK-8 and Transwell experiments showed that the inhibitor of miR-486-5p notably attenuated the inhibition of cell proliferation and metastatic capabilities mediated by EXOs (Fig. 4B-D). Furthermore, co-treatment with the miR-486-5p inhibitor and EXOs led to higher levels of glucose consumption and lactate production in the cells, along with elevated ECAR levels (Fig. 4E-G). Additionally, the inhibitor promoted cell stemness, as evidenced by the enhanced sphere-forming ability (Fig. 4H). These findings provide compelling evidence regarding the effect of miR-486-5p on EXO-mediated suppression of CRC cell stemness.

Fig. 4.

Fig. 4

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The miR-486-5p inhibits the proliferation, migration, glycolysis, and stemness of CRC cells. The miR-486-5p-inhibitor was introduced into CRC cells. Then, miR-486-5p-inhibitor cells were cultured with or without hUC-MSC-EXOs. (A) Using qRT-PCR, the level of miR-486-5p was investigated. (B) The CCK-8 assay was employed to gauge the CRC cells’ ability to proliferate. (C–D) The Transwell test was utilized to identify the cells’ capacity for migration and invasion. (E-G) Glucose consumption, lactic acid production, and the level of ECAR were detected by kits. (H) The sphere formation ability was tested in CRC cells. * represents P < 0.05 compared with the Control or EXO group

Mir-486-5p regulates the transcription of NEK2

To study the target genes of miR-486-5p, upregulated DEmRNAs in CRC cells were obtained from the GSE202408 dataset. Subsequently, the starBase, miRDB and mirDIP 3 databases were used to predict the target genes of miR-486-5p. A Venn diagram was plotted and 4 upregulated DEmRNAs (NEK2, Phosphatidylinositol 3-Kinase Regulatory Subunit Alpha (PI3KR1), BTB domain containing 3 (BTBD3), Folliculin interacting protein 2 (FNIP2)) targeting binding sites with miR-486-5p were obtained (Fig. 5A). Among these, NEK2 showed the highest differential expression in CRC cells (Fig. 5B). As illustrated in Fig. 5C, miR-486-5p directly bound to the NEK2 3’-UTR. To delve deeper into the underlying molecular mechanisms, CRC cells overexpressing miR-486-5p were generated. The expression of miR-486-5p was markedly elevated by the miR-486-5p mimic (Fig. 5D). Importantly, it was shown that increased miR-486-5p levels suppressed NEK2 expression (Fig. 5E-F). More importantly, RIP results revealed a notable alteration in NEK2 levels in cells overexpressing miR-486-5p (Fig. 5G). Figure 5H depicted the luciferase assay results, which revealed a significant decrease in the luciferase activity of the NEK2 wild-type compared to the NEK2 mutant in HEK293 cells overexpressing miR-486-5p. This suggested a connection between NEK2 and miR-486-5p.

Fig. 5.

Fig. 5

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miR-486-5p modulates the transcription of NEK2. (A) Venn diagram of possible target mRNAs of miR-486-5p and up-regulated DEmRNAs. (B) Correlation analysis between miR-486-5p and 4 mRNAs. (C) Box plot of the expression levels of candidate target genes NEK2, PI3KR1, BTBD3, and FNIP2 in the normal cells group and CRC cells group. (D) The levels of miR-486-5p were examined in CRC cells transfected with miR-486-5p by qRT-PCR. (E) The levels of NEK2 were identified in overexpressing miR-486-5p CRC cells. (F) The protein level of NEK2 was measured in CRC cells with overexpressing miR-486-5p by western blot. (G) RIP was applied to confirm the interaction between miR-486-5p and NEK2 in overexpressed miR-486-5p CRC cells. (H) By using the dual luciferase assay on HEK293 co-transfected with NEK2 and miR-486-5p, the activity of NEK2 was confirmed. ns represents no difference compared with the NC mimic group; * represents P < 0.05 compared with the NC mimic group

miR-486-5p regulates the proliferation, metastatic, glycolysis, and stemness of CRC cells by suppressing the transcription of NEK2

To investigate the role of NEK2 in CRC cells, the NEK2 overexpressed CRC cells were generated. As shown in Fig. 6A, there were increased level of miR-486-5p by the miR-486-5p mimic, yet there was no significant change in the miR-486-5p mimic + oe-NEK2 group. Moreover, cells transfected with the NEK2 plasmid showed increased NEK2 levels, while the miR-486-5p mimic significantly reduced NEK2 expression (Fig. 6B). Cellular experiments demonstrated that the proliferation capability of cells was enhanced in NEK2 overexpressed cells, and the effect was reversed by the miR-486-5p mimic (Fig. 6C). Similarly, the migration and invasion capabilities of the CRC cells exhibited comparable trends (Fig. 6D-E). Importantly, in CRC cells, the miR-486-5p mimic inhibited the increase in glucose consumption, lactate production, and ECAR levels induced by NEK2 overexpression, indicating a reduction in cellular glycolytic capacity (Fig. 6F-H). The increased sphere-forming ability of CRC cells caused by NEK2 overexpression also suggests that NEK2 plays a pro-cancer role, whereas the overexpression of miR-486-5p inhibits cancer cell stemness by binding to NEK2 (Fig. 6I). In conclusion, these data indicate that EXOs inhibit the malignant phenotype and stemness of CRC cells by secreting miR-486-5p, and the mechanism by which miR-486-5p inhibits the expression of NEK2 and glycolytic capacity of CRC cells.

Fig. 6.

Fig. 6

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miR-486-5p influences the growth, migration, glycolysis, and stemness of CRC cells by blocking NEK2 transcription. The miR-486-5p mimic and overexpressed NEK2 plasmid were introduced into CRC cells. (A) The levels of miR-486-5p were investigated. (B) The levels of NEK2 were examined. (C) The proliferation capacity of CRC cells was assessed utilizing CCK-8 experiment. (D-E) Using a Transwell test, the migratory and invasion capabilities of the cells were identified. (F-H) Glucose consumption, lactic acid production, and the level of ECAR were detected by kits. (I) The sphere formation ability was tested in CRC cells. * represents P < 0.05 compared with the Control or miR-486-5p-mimic + oe-NC group

Discussion

CRC is the fourth most common cancer and has attracted considerable attention [3]. Research has shown that the death rate of CRC is age dependent. Interestingly, the incidence of colon cancer is becoming more common in younger patients. Research has recommended lowering the CRC risk screening age to 45 years [17]. However, it is concerning that about 50% of CRC patients will develop colorectal metastases, especially unresectable liver metastases with a poor prognosis [18, 19]. Currently, chemotherapy is the main method used to treat unresectable metastatic disease, and the risk of side effects is high. Therefore, there is an urgent need to develop novel treatments.

In recent decades, EXOs have been identified as an effective treatment for several types of cancer. Among several EXOs, hUC-MSC-EXOs have the advantages of easy culturing, self-renewal, and immunosuppression [20]. hUC-MSC-EXOs, as a novel “cell-free therapy, ” are widely applied in cancer by releasing proteins, RNAs, and lipids [21]. Research has shown that hUC-MSC-EXOs have immense potential for the treatment of acute lung injury, as they could reduce oxidative stress and inflammatory responses both in vivo and in vitro [22]. Moreover, hUC-MSC-EXOs exert an inhibitory effect on hepatomas and wild-type tumors [11, 23]. Consistently, it has been discovered that CRC cells can absorb hUC-MSC-EXOs, allowing them to exert their regulatory effects, which is the same as in previous studies. Furthermore, after co-culturing with CRC cells, hUC-MSC-EXOs displayed an inhibitory effect on the growth and migration abilities of CRC cells, while also regulating metabolic processes (reducing glycolytic capacity) and cell stemness within CRC cells. These results reveal that hUC-MSC-EXOs could applied in CRC as an effective treatment for CRC.

miRNAs, which are secreted components of EXOs, have been demonstrated to regulate cancer cell progression and various physiological activities [24]. Several studies have shown that MSC-EXOs can inhibit the growth and metastasis of CRC cells through miR-100, miR-16-5p, miR-375, and other mechanisms. Jahangiri et al. demonstrated the high expression of miR-100 and miR-143 in BM-MSC-EXOs. Importantly, BM-MSC-EXOs facilitated apoptosis of SW480 cells through the miR-100/mTOR/miR-143 axis [25]. The therapeutic effect of BM-MSC-EXOs on CRC is also related to miR-16-5p, which suppresses ITGA2 levels and impedes the progression of CRC [26]. Zaharie et al. confirmed that the overexpression of miR-375 in EXOs isolated from CRC patients could significantly restrict CRC cell proliferation via the Bcl-2 pathway [27]. In addition, exosomal miR-3940-5p derived from hUC-MSCs directly binds to ITGA6, inducing the suppression of EMT in CRC cells, which is associated with the inactivation of the TGF-β1 pathway [28]. In our study, we identified that miR-486-5p, secreted by hUC-MSC-EXOs, may regulate the malignant phenotype of CRC cells, consistent with findings from previous studies.

Research has highlighted miR-486-5p as a promising biomarker for cancer diagnosis, prognosis, and therapeutic targeting across various cancer types. Interestingly, its low expression in osteosarcoma suggests an inherent anticancer function [29]. It also inhibits lung cancer progression by targeting CD133, which impairs the PI3K/AKT signaling pathway [30]. Furthermore, miR-486-5p has been strongly linked to CRC in significant ways [31, 32]. By targeting PIK3R1, miR-486-5p effectively suppresses the invasion and migration of CRC cells [13] and the growth of CRC by regulating neuropilin-2 [12]. In this study, high miR-486-5p expression in hUC-MSC-EXOs entering CRC cells inhibited cell proliferation, migration, glycolysis, and stemness. It is important to highlight that miRNAs commonly combine with the 3’UTR region of target mRNAs to modulate intracellular signaling pathways. Following analysis of miR-486-5p’s downstream targets, we observed substantial alterations in NEK2 expression in CRC cells treated with hUC-MSC-EXOs. Experimental validation confirmed the interaction between miR-486-5p and NEK2. By inhibiting NEK2 transcription, miR-486-5p effectively modulated the proliferation, migration, glycolysis, and stemness of CRC cells. This indicated that hUC-MSC-EXOs suppressed CRC progression through the miR-486-5p/NEK2 axis.

NEK2, which belongs to the Nek family of serine/threonine kinases, has been implicated in CRC. A previous study showed that miR-128 binds to the 3’-UTR of NEK2, causing attenuation of CRC cell cycle progression and proliferation [33]. NEK2 affects mitotic progression and stemness maintenance, and can cooperate with ERK to promote proliferation and stemness in CRC [16]. Consistent results were observed in our study, with a significant suppression of NEK2 caused by miR-486-5p.

While our research has identified that miR-486-5p suppresses NEK2 transcription, the downstream signaling pathways remain unclear. Additionally, additional validation of the therapeutic efficacy of EXO-secreted miR-486-5p should be performed in animal models.

Conclusion

In summary, our initial findings demonstrated that hUC-MSC-EXOs can enter CRC cells and effectively impede their proliferation, migration, glycolysis, and stemness. Further analysis revealed significant upregulation of miR-486-5p in EXOs and cells co-cultured with EXOs. Mechanistically, the inhibitory effect of hUC-MSC-EXO-delivered miR-486-5p on CRC progression was attributed to suppression of NEK2 transcription. These findings offer compelling support for the potential therapeutic utility of hUC-MSC-EXOs in CRC management.

Acknowledgements

Not applicable.

Author contributions

Conception and design: F. C, Y. C and X. W. Data analysis and interpretation: Y. C and X. W. Manuscript writing: F. C, Y. C and W. Z. All authors reviewed the manuscript .

Funding

Not applicable.

Data availability

The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.

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