Human normal colorectal mucosa NCM460 cell line (cat no. SNL-519; SUNNCELL Co., Ltd.), four colon cancer cell lines, namely HCT116 (cat no. CCL-247EMT), SW480 (cat no. CCL-228), SW620 (cat no. CCL-227) and Caco-2 (cat no. HTB-37), and 293T (cat no. CRL-3216) cells were purchased from American Type Culture Collection. These cell lines were cultured in high-glucose DMEM (Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA) in a 37°C incubator with 5% CO₂.

Between January 2020 and July 2021, 50 CRC tissues and associated normal tissues were obtained from the Department of Colorectal Surgery, Zhejiang Provincial People's Hospital (Hangzhou, China). Before surgery, neither radiation nor chemotherapy had been administered to any of the patients. The adjacent normal tissues that were >5 cm from the tumor's margin were received. The present study was approved (approval no. 2020059) by the ethical committee of the Zhejiang Provincial People's Hospital (Hangzhou, China). Every participant signed a statement of informed consent. Clinicopathological characteristics of patients with CRC (including sex and age distribution) are presented in Table I.

The microRNA data (accession nos. GSE183437 and GSE156719) were downloaded from GEO databases in NCBI (23). Microarray data of GSE183437 was on account of GPL16791 Platform Illumina HiSeq 2500 (Homo sapiens), which included 5 CRC tissues and 5 non-cancerous tumor-adjacent tissues, while GSE156719 was on account of GPL20712 Platform Agilent-070156 human miRNA [miRNA version], which included 3 pairs of colorectal tumor tissues and normal tissues. Differentially expressed miRNAs (DEmiRNAs) were identified through GEO2R online platform (https://www.ncbi.nlm.nih.gov/geo/geo2r/), which is a widely used tool that can be used to analyze data from any GEO series and significance analysis of microarray (SAM), to determine the differential expression of miRNAs among groups. miRNAs were considered to be differentially expressed according to the P<0.05 threshold from the limma analysis and median false discovery rate <0.05 from SAM. miRs with the top 46 differences were selected for heat mapping using GeneSpring GX statistical software (version 7.3; Agilent Technologies, Inc.).

The total RNA of the cultured cells and the tissues was extracted using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.), and total RNA was reverse-transcribed to cDNA using the iScript advanced cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. RT-qPCR was performed using SYBR^®PrimeScript™ RT-PCR kit (Takara Bio, Inc.) on the Applied Biosystems Quantstudio6 flex (Applied Biosystems; Thermo Fisher Scientific, Inc.). For detection of microRNAs, a universal reverse primer that is complementary to a sequence within the RT stem-loop primer is 5′-GCAGGGTCCGAGGTATTC-3′. The specific forward primers for microRNAs were as follows: miR-25 forward, 5′-CATTGCACTTGTCTCGGT-3′; miR-18a forward, 5′-ACTGCCCTAAGTGCTCCT-3′; miR-92a forward, 5′-AATTATTGCACTTGTCCC-3′; miR-203 forward, 5′-GTGAAATGTTTAGGACCA-3′; miR-143 forward, 5′-TGAGATGAAGCACTGTAG-3′; miR-375 forward, 5′-TTTGTTCGTTCGGCTCGC-3′; miR-29a forward, 5′-TAGCACCATCTGAAATCG-3′; miR-137 forward, 5′-TTATTGCTTAAGAATACG-3′. The other qPCR primers used in the present study were as follows: FBWX7 forward, 5′-CACTCAAAGTGTGGAATGCAGAGAC-3′ and reverse, 5′-GCATCTCGAGAACCGCTAACAA-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; and GAPDH forward, 5′-TCAACGACCCCTTCATTGACC-3′ and reverse, 5′-CTTCCCGTTGATGACAAGCTTC-3′. The thermocycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The fold changes were calculated using the 2^−∆∆Cq method (24).

When HCT116 and Caco-2 cells (5×10⁵ cells/well) in six-well plates reached ~80% confluence, miR-25-3p mimics (20 nM), mimics negative control (mimics NC, 20 nM), miR-25-3p inhibitor (20 nM), inhibitor NC (20 nM), small interfering (si) FBXW7 (50 nM) and si-Scramble (50 nM) were transfected into cells at 37°C for 24 h using Lipofectamine^®2000 according to manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). siRNA, miR mimics and miR inhibitors were provided by GenScript (Nanjing) Co., Ltd. The sequences are as follows: miR-25-3p mimics, 5′-CAUUGCACUUGUCUCGGUCUGA-3′; mimics NC, 5′-CUGUAACUGCUCUGCUGUCGUA-3′; miR-25-3p inhibitor, 5′-UCAGACCGAGACAAGUGCAAUG-3′; inhibitor NC, 5′-UAGAUAGUGACGAGAAACCCCG-3′; siFBXW7, 5′-CAAUUGUGUAGACGAUAUACU-3′; si-Scramble, 5′-UAUAUUCUCAUGAACAGGAUG-3′.

The FBXW7 gene was amplified by PCR utilizing human cDNA extracted from adjacent normal tissues. The amplified fragments were cloned into the pcDNA3.1(+) plasmid (Shanghai GenePharma Co., Ltd.), named pcDNA3.1-FBXW7. Transfection was conducted using Lipofectamine^®2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

RT-qPCR analysis and western blotting were used to detect the successful knockdown or upregulation of miR-25-3p and FBXW7 expression, respectively.

The proliferation of HCT116 and Caco-2 cells was measured using a Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Inc.). After 24, 48 and 72 h post-transfection, CCK-8 reagent (10 µl/well) was added to HCT116 and Caco-2 (1×10⁶ cells/well), and then incubated at 37°C with 5% CO₂ for another 3 h. Subsequently, cell proliferation was analyzed at 450 nm, using a microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, Inc.).

HCT116 and Caco-2 cells (5×10⁵ cells) were seeded in 6-well plates overnight at 37°C. Apoptotic rates were determined using the Annexin V-FITC Apoptosis Detection Kit (cat. no. ab14082; Abcam) 48 h post-transfection. Cells were digested with 0.25% trypsin (Millipore Sigma; Merck KGaA), centrifuged at 300 × g for 5 min at 4°C, resuspended in 20 µl binding buffer, and incubated with 5 µl Annexin V-FITC and 1 µl Propidium Iodide (PI) in a dark room at room temperature for 20 min. The stained cells were then analyzed using BD FACSCalibur Flow Cytometer System (BD Biosciences). Data analysis was performed using BD FACSuite™ software (Version 6.0; BD Biosciences). The results demonstrated healthy viable cells in the lower left quadrant on the scatter plot as (FITC⁻/PI⁻). The lower right quadrant (Q3) represented the early-stage apoptotic cells as (FITC⁺/PI⁻). The upper right quadrant (Q2) represented late-stage apoptotic cells as (FITC⁺/PI⁺). The calculation was made as follows: Apoptotic rate=percentage of early-stage apoptotic cells (Q3) + percentage of late-stage apoptotic cells (Q2).

Caspase-3 activity assay was performed in HCT116 and Caco-2 cells using a Caspase-3 colorimetric assay kit (cat no. 556485; BD Biosciences) according to the manufacturer's protocol.

Using the online bioinformatics tools TargetScan 7.0 (http://www.targetscan.org/vert_70/) and miRanda (v3.3a; http://www.microrna.org), the binding sites between miR-25-3p and FBXW7 were predicted. The FBXW7 3′-UTR partial sequence, which contains the binding site or mutated binding site, was generated and inserted into a dual-luciferase pGL3 plasmid (Promega Corporation) by GenScript (Nanjing) Co., Ltd. Using Lipofectamine^®2000 (Invitrogen; Thermo Fisher Scientific, Inc.), a total of 200 ng of either wild-type (wt)-FBXW7-3′UTR-pGL3 or mutant (mut)-FBXW7-3′UTR-pGL3 reporter plasmids were co-transfected with 40 nM miR-25-3p mimics, mimics NC, miR-25-3p inhibitor, and inhibitor NC into 293T cells. Luciferase assays were performed 48 h post transfection using the Dual-luciferase^® Reporter Assay System (Promega Corporation). The luciferase activity was normalized to Renilla luciferase activity.

Total protein was extracted from cells using the RIPA lysis buffer 48 h after transfection (Beyotime Institute of Biotechnology). A BCA protein assay reagent kit was used to measure the protein concentration. Proteins (25 µg/lane) were separated by SDS-PAGE using 8% gels and then electroblotted on PVDF membranes. The membranes were blocked by soaking in 5% skimmed milk for 1 h at 4°C overnight. Specific primary antibodies were used to stain the membranes at 4°C overnight, followed by the corresponding secondary antibody at room temperature for 1 h. The enhanced chemiluminescence (ECL) detection system (Pierce; Thermo Fisher Scientific, Inc.) was used to identify protein bands, and ImageJ software (version 1.46; National Institutes of Health) was used to quantify them. The specific antibodies used in the present study were as follows: FBXW7 (1:200; cat. no. ab105752; Abcam), c-Myc (Y69; 1:1,000; cat no. ab32072; Abcam), Notch 2 (1:1,000; cat. no. ab118824; Abcam), YAP (1:1,000; cat. no. ab52771; Abcam), cyclin E (1:1,000; cat. no. ab33911; Abcam), β-actin (1:1,000; cat no. ab8277; Abcam) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat no. ab6721; Abcam).

SPSS 21.0 software (IBM Corp.) was used to conduct the statistical analysis. The data are presented as the mean ± standard deviation. One-way ANOVA was used to analyze comparisons between various groups, and Tukey's post hoc test was used to confirm the results. Using the unpaired Student's t-test, two groups were compared. The correlation between miR-25-3p and clinicopathological features of patients with CRC was analyzed using the chi-square test and Fisher's exact test. The correlation coefficient between miR-25-3p and FBXW7 was calculated using Spearman's rank correlation coefficient. The Kaplan-Meier method was used to create the survival curves, and the log-rank test was used to compare them statistically. The threshold for statistical significance was set at P<0.05.

To delve into the function of miRNAs in the context of CRC, an analysis of DEmiRNAs from two GEO datasets was conducted (GSE183437 and GSE156719) using the GEO2R online platform (https://www.ncbi.nlm.nih.gov/geo/geo2r/). The present analysis revealed a total of 46 dysregulated miRNAs in CRC tissues, with 20 miRNAs exhibiting increased expression and 26 showing decreased expression relative to the adjacent normal tissues (Fig. 1A). Subsequently, further validation of the five most significantly upregulated miRNAs (miR-18a, miR-92a, miR-25, miR-203 and miR-143) and the three most notably downregulated miRNAs (miR-375, miR-29a and miR-137) was performed across the two GEO datasets. The significantly DEmiRNAs, which were consistent with previous results (25–31), were presented in Fig. 1B, indicating the reliability of the screening results obtained by the microarray analysis. Of these miRNAs, the expression of miR-25-3p was found to be the most significantly increased in the CRC tissue. miR-25-3p has previously been linked to tumorigenesis of CRC (32). By contrast, little is known about the roles of miR-25-3p for CRC. It was therefore decided to focus on miR-25-3p for further molecular analyses, to clarify the previously unknown role of miR-25-3p in CRC.

In order to investigate the relevance of miR-25-3p in CRC, 50 pairs of CRC tissues and adjacent normal tissues were utilized for the validation of its aberrant expression. RT-qPCR analysis confirmed a significant upregulation of miR-25-3p in the CRC tissues as opposed to the adjacent normal tissues. This observation was in alignment with the differential expression patterns observed in the GEO datasets, revealing a consistently differential expression with the GEO datasets (Fig. 1C). Furthermore, the relationship between miR-25-3p expression and clinicopathological features was also analyzed. The samples were divided into high and low miRNA expression groups according to the median expression value as the cutoff point. It was found that high expression of miR-25-3p was closely associated with tumor size, distant metastasis and tumor-node-metastasis stage (Table I). Furthermore, the association between miR-25-3p expression and overall survival rate in CRC was investigated, and the overall survival rate of patients with CRC with high miR-25-3p expression was significantly shorter compared with low miR-25-3p expression (Fig. 1D). Therefore, the findings revealed that miR-25-3p might act as an oncogene in the development of CRC.

Taken a step further, to validate whether the altered expression of miR-25-3p was also present in CRC cell lines, the expression of miR-25-3p was detected in HCT116, SW480, SW620 and Caco-2 cell lines and human normal colorectal mucosa NCM460 cell line was used as a control. It was found that miR-25-3p expression was significantly upregulated in CRC cells compared with NCM460 cells, which exhibited the same trend as the results in CRC tissues (Fig. 2A).

To determine the effect of miR-25-3p on CRC cell proliferation, HCT116 and Caco-2 cells were transfected with miR-25-3p inhibitor. Post-transfection with the miR-25-3p inhibitor, a significant reduction in miR-25-3p levels was observed in both HCT116 and Caco-2 cells, as depicted in Fig. 2B. Moreover, the suppression of miR-25-3p led to a significant decrease in cell proliferation and a concurrent increase in the rate of apoptosis in HCT116 and Caco-2 cells when compared with the NC group (Fig. 2C-E). Collectively, these results revealed that knockdown of miR-25-3p suppressed CRC cell proliferation and promoted cell apoptosis.

To further explore the possible mechanisms of miR-25-3p in CRC cells, potential target genes of miR-25-3p were searched using TargetScan and miRanda databases. FBXW7 was chosen as a target gene of miR-25-3p in the present study (Fig. 3A). RT-qPCR assay confirmed the successful overexpression or knockdown of miR-25-3p after transfection of miR-25-3p mimics or inhibitor, respectively, as demonstrated in Fig. 3B. As it has been previously reported, FBXW7 is a tumor suppressor with a role in various types of human cancers, including CRC (33–35). Additionally, a luciferase reporter assay was conducted to confirm the interaction between miR-25-3p and the 3′UTR of FBXW7. As illustrated in Fig. 3C, the overexpression of miR-25-3p led to a significant decrease in luciferase activity when the wt-FBXW7 3′UTR was present, whereas the activity of the mut-FBXW7 3′UTR remained unaltered in 293T cells. Conversely, the knockdown of miR-25-3p resulted in an increase in luciferase activity for the wt-FBXW7 3′UTR. Moreover, the overexpression of miR-25-3p in HCT116 and Caco-2 cells led to a significant reduction in both mRNA and protein levels of FBXW7 (Fig. 3D and E), whereas the knockdown of miR-25-3p yielded the opposite effect in mRNA level as shown in Fig. 3D. These results collectively suggested a direct and functional interaction between miR-25-3p and FBXW7. Next, the relationship between miR-25-3p and FBXW7 in CRC tissues was investigated. First, the level of FBXW7 mRNA in four CRC cell lines and 50 patients with CRC was measured by RT-qPCR. The results of RT-qPCR showed that FBXW7 was significantly decreased in the CRC cells and tissues compared with NCM460 cells and the adjacent normal tissues, respectively (Fig. 3F and G). Furthermore, as revealed in Fig. 3H, there was an inverse correlation between miR-25-3p and FBXW7 expression levels in the 50 patients with CRC (r=−0.7989; P<0.01). These data demonstrated that FBXW7 may be a functional target of miR-25-3p in CRC.

To elucidate the functional role of FBXW7 in CRC, FBXW7 was overexpressed in CRC cell lines and the impact on cell proliferation and apoptosis was subsequently assessed. For this purpose, plasmids named pcDNA-FBXW7 were constructed and introduced into HCT116 and Caco-2 cells. Western blot analysis confirmed the successful overexpression of FBXW7 following the introduction of the pcDNA-FBXW7 plasmids, as depicted in Fig. 4A. The overexpression of FBXW7 led to a significant reduction of cell proliferation in both HCT116 and Caco-2 cells, as determined by cell proliferation assays, compared with the control group (Fig. 4B and C). Moreover, the apoptotic rate in cells overexpressing FBXW7 was significantly higher than that observed in the control group (Fig. 4D), underscoring the potential of FBXW7 as a tumor suppressor in CRC. Overall, the present findings revealed that FBXW7 overexpression inhibited CRC cell proliferation and enhanced cell apoptosis.

It was anticipated that miR-25-3p targets FBXW7 in colon cancer cells to have antitumor effects in light of the aforementioned results showing that overexpression of FBXW7 decreased CRC cell proliferation and enhanced cell apoptosis. HCT116 and Caco-2 cells received simultaneous transfections of si-FBXW7 and miR-25-3p inhibitor. Western blot assay results identified that FBXW7 was successfully knocked down by the si-FBXW7 (Fig. 5A). Then, apoptosis and proliferation of CRC cells were investigated. The findings demonstrated that miR-25-3p inhibitor-transfected HCT116 and Caco-2 cells showed reduced cell proliferation when compared with inhibitor NC-transfected cells; however, these effects were partially diminished by si-FBXW7 (Fig. 5B and C). In the caspase 3 activity assay, HCT116 and Caco-2 cells treated with the miR-25-3p inhibitor exhibited increased caspase 3 activity compared with cells treated with the NC inhibitor. However, the silencing of FBXW7 with si-FBXW7 negated the enhanced caspase 3 activity observed with miR-25-3p inhibition (Fig. 5D and E). A similar pattern of apoptotic effects was observed in HCT116 and Caco-2 cells following transfection with the miR-25-3p inhibitor, which were counteracted by the concurrent knockdown of FBXW7 using si-FBXW7 (Fig. 5F and G). Collectively, the present results indicated that the pro-apoptotic effects of miR-25-3p inhibition on CRC cells are partially mitigated by the suppression of FBXW7.

It has been revealed that FBXW7 is responsible for the binding to and the degradation of several oncogenes, including cyclin E, Yap, Notch and c-Myc, and thus inhibits tumor cell proliferation and invasion, to exert its tumor-suppressive effects (36–39). The FBXW7-related oncogenic proteins were detected: cyclin E, Yap, Notch and c-Myc. Compared with the inhibitor NC group, it was discovered that miR-25-3p knockdown significantly reduced the expression of cyclin E, Yap, Notch and c-Myc. However, these inhibitory effects were reversed by si-FBXW7 in HCT116 and Caco-2 cells (Fig. 6A-C). All of these findings pointed to the possibility that miR-25-3p functions as an oncogene in CRC cells by suppressing FBXW7 and subsequently indirectly promoting these oncoproteins.