The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) database (http://ualcan.path.uab.edu) [24, 25] was utilized to compare the expression levels of CELF6 between colon adenocarcinoma (COAD) tissues and paired malignant non-transformed tissues. The prognostic significance of CELF6 expression in CRC was assessed using the PrognoScan database (http://dna00.bio.kyutech.ac.jp/PrognoScan/index.html) [26]. A significance threshold of p 0.05 was applied in both databases.

Tissue samples were collected from sixty CRC patients, along with matching paracancerous normal tissues. Immediately following excision, all tissue samples were preserved at –80 °C. The study received approval from the Ethics Committee of Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University (Approval No. Med-Eth-Re [2023] 33). Written informed consent was obtained from the patients or their families/legal guardians.

Five human CRC cell lines (HT29, HCT116, LoVo, SW48, and HCT15) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The normal colonic epithelial cell line NCM460 was sourced from the BeNa Culture Collection (Beijing, China). All cell lines were authenticated by short tandem repeat (STR) profiling and were confirmed to be free of mycoplasma contamination. Cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a 5% CO₂ incubator.

The CELF6 cDNA sequence, cloned into the pcDNA vector, and a control vector were provided by GenePharma (Shanghai, China). Additionally, short hairpin RNAs (shRNAs) targeting CELF6 and HOXA5 were also supplied (shNC: 5^′-TTCTCCGAACGTGTCACGTAA-3^′; shCELF6-1#: 5^′-GCCCAGATTTACTTCTTTCAA-3^′; shCELF6-2#: 5^′-CATACAGACATTCCTGCCCTT-3^′; shHOXA5: 5^′-GCCATTATAGCGCCTGTATAA-3^′). The HCT15 and SW48 cell lines were transfected with these constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Total RNA was extracted from tissue samples and cultured cells using TRIzol reagent (Invitrogen, USA). The RNA was then reverse-transcribed into complementary DNA (cDNA) using the TaqMan™ Reverse Transcription Reagent (Invitrogen, USA). qRT-qPCR was performed using the ABI 7500 Fast Real-Time PCR System. The following primers were used: CELF6, forward: 5^′-CCCATCGGGGTCAATGGATTC-3^′, reverse: 5^′-GCCCGTTATTGTAGAGCGTGT-3^′; HOXA5, forward: 5^′-AACTCATTTTGCGGTCGCTAT-3^′, reverse: 5^′-TCCCTGAATTGCTCGCTCAC-3^′; GAPDH, forward: 5^′-GGAGCGAGATCCCTCCAAAAT-3^′, reverse: 5^′-GGCTGTTGTCATACTTCTCATGG-3^′.

Total protein was extracted from cells and tissues using radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Shanghai, China), and the protein samples were electrophoresized and transferred to polyvinylidene fluoride (PVDF) membranes at 4 °C. The membranes were then blocked with 5% skimmed milk at room temperature. Primary antibodies were applied at 4 °C: anti-CELF6 (ab173282; Abcam, Shanghai, China; 1/1000), anti-HOXA5 (ab140636; Abcam, China; 1/1000), anti-cyclin-dependent kinase 1 (CDK1) (ab133327; Abcam, China; 1/10,000), anti-cyclin D1 (ab16663; Abcam, China; 1/1000), anti-cyclin B1 (ab32053; Abcam, China; 1/50,000), anti-p27^kip1 (ab32034; Abcam, China; 1/5000), anti-Nanog (ab109250; Abcam, China; 1/2000), anti-OCT4 (ab200834; Abcam, China; 1/10,000), anti-SRY-box transcription factor 2 (SOX2) (ab92494; Abcam, China; 1/1000), anti-CD133 (ab222782; Abcam, China; 1/2000), and anti-$\beta$-actin (ab8227; Abcam, China; 1/1000). Membranes were incubated with goat anti-rabbit immunoglobulin G (IgG) secondary antibodies (ab97051; Abcam, China) at dilutions of 1/2000 or 1/20,000. After adding chemiluminescent substrates from Invitrogen to visualize the protein bands, images were captured, and grayscale values were quantified using ImageJ software, version 1.53 (NIH, Bethesda, MD, USA).

Tissue sections were fixed in 4% formaldehyde, embedded in paraffin, and subjected to antigen retrieval. Sections (5 µm) were incubated with the following primary antibodies at room temperature: anti-CELF6 rabbit monoclonal antibody (Abcam, China; ab173282; 1/50 dilution), anti-HOXA5 rabbit polyclonal antibody (Invitrogen, USA; PA5-69008; 5.0 µg/mL dilution), anti-SOX2 rabbit monoclonal antibody (Abcam, China; ab92494; 1/1000 dilution), and anti-CD133 rabbit monoclonal antibody (Abcam, China; ab222782; 1/1000 dilution). Biotinylated secondary antibodies were then applied, and the sections were visualized using a microscope after staining with 3,3’-diaminobenzidine (DAB) chromogenic agents.

Cell viability was assessed using the cell counting kit-8 (CCK-8) assay (Beyotime Biotechnology, Shanghai, China). Cells were plated into 96-well plates at a density of 2000 cells per well and incubated for 0, 24, 48, or 72 hours. Following incubation, 10 µL of CCK-8 solution was added to each well, and absorbance was measured at 450 nm using a microplate reader. For the colony formation assay, the cells were seeded at a density of 1000 cells per well in 6-well plates. After two weeks of incubation, colonies were fixed, stained, and counted under a microscope (Olympus, Tokyo, Japan).

Cell cycle distribution was analyzed using the Cell Cycle and Apoptosis Analysis Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Aldehyde dehydrogenase 1 (ALDH1)-PE (#65583; 1/50 dilution) and CD44-allophycocyanin (APC) (#80813; 1/160 dilution) antibodies were used to identify ALDH1- or CD44-positive cells. FCM was conducted using a flow cytometer (Beckman Coulter, Fullerton, CA, USA) after staining.

The cells were collected and prepared as a single-cell suspension in a medium supplemented with basic fibroblast growth factor (20 ng/mL), recombinant human epidermal growth factor (10 ng/mL), and B27 (2%). Sphere formation was assessed after 14 days when spheres reached a diameter of 50 µm.

The stability of HOXA5 mRNA was evaluated using an ActD assay. The cells, either transfected with CELF6 overexpression plasmids or control cells, were treated with ActD (5 µg/mL; Sigma-Aldrich, St. Louis, MO, USA). HOXA5 mRNA levels were measured by qRT-PCR at various time points (0, 2, 4, and 8 hours).

The wild-type or mutant HOXA5 3^′ UTR sequences were cloned into luciferase reporter vectors. These vectors were co-transfected with CELF6 overexpression plasmids, and luciferase activity was measured using the Dual Luciferase Assay Kit (Thermo Scientific, China).

The RIP assay was performed using the Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen, USA). Cell lysates were incubated with anti-CELF6 (5 µg) or IgG-conjugated magnetic beads. The RNA-protein complexes were then isolated and analyzed by qPCR.

The cell lysates were incubated with 50 pmol of a biotin-labeled HOXA5-RNA probe in an RNA binding buffer at 4 °C for 2.5 hours. RNA pull-down assays were performed using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific, China). After incubation, the complexes were captured with pre-saturated streptavidin beads for 30 minutes, washed, and then subjected to western blot analysis.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University (Approval No. Med-Eth-Re [2023] 33). CELF6 overexpressing or control SW48 cells (5 $\times$ 10⁵) were subcutaneously injected into the right flank of 4-week-old male BALB/c nude mice (n = 6 per group) to develop xenografts following anesthesia with 3% isoflurane. Tumor volume was calculated using the formula: length $\times$ width²/2. At the end of the in vivo experiment, the mice were euthanized by cervical dislocation and the xenograft tissues were collected for qRT-PCR and IHC analysis.

Data are expressed as mean $\pm$ standard deviation (SD). Statistical analyses were performed using Student’s t-test, paired t-test or ANOVA with GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). A p-value of 0.05 was considered statistically significant.

The investigation began with an analysis of CELF6 expression in colon adenocarcinoma (COAD) using the UALCAN database. This analysis revealed that CELF6 expression was significantly reduced in COAD tissues compared to healthy controls (Fig. 1A). To further explore the clinical relevance of CELF6, we examined its association with survival outcomes in CRC patients through the PrognoScan database. Our analysis indicated that lower CELF6 levels were correlated with poorer survival rates, including overall survival (Cox P = 0.039360) and disease-specific survival (Cox P = 0.081047) (Fig. 1B).

Fig. 1.

Cytosine-uridine-guanine-binding protein (CUG-BP), Elav-like family (CELF) 6 is downregulated in colorectal cancer (CRC) tissues and cells. (A) Analysis of CELF6 expression in colon adenocarcinoma (COAD) and adjacent non-transformed tissues using data from The Cancer Genome Atlas (TCGA) database accessed via UALCAN. (B) Kaplan-Meier survival curves comparing high and low CELF6 expression levels in CRC patients, as retrieved from the PrognoScan database. (C) quantitative real-time polymerase chain reaction (qRT-PCR) analysis of CELF6 mRNA expression in 60 CRC tissues and corresponding adjacent non-cancerous tissues (unpaired, left; paired, right). (D) Immunoblotting analysis of CELF6 protein expression in four randomly selected pairs of CRC and adjacent non-cancerous tissues. (E) Representative immunohistochemistry (IHC) images and statistical results showing CELF6 expression in two pairs of CRC and adjacent non-transformed tissues at 100$\times$ and 200$\times$ magnifications. Scale bars, 100µm or 200µm. (F,G) qRT-PCR and western blot analysis of CELF6 mRNA and protein expression in CRC cell lines and the NCM460 cell line. The results are presented as mean $\pm$ SD of three independent experiments (n = 3). Statistical significance was determined by comparisons with the normal group or the NCM460 group (**p 0.01; ***p 0.001).

Next, we validated these findings in a cohort of 60 CRC tumor specimens and their matched adjacent non-cancerous tissues. qRT-PCR analysis demonstrated a significant reduction in CELF6 mRNA expression in CRC tissues compared to normal tissues (Fig. 1C). Western blotting and immunohistochemistry (IHC) further confirmed the downregulation of CELF6 protein in CRC tissues (Fig. 1D,E). Similar trends were observed in CRC cell lines, where both mRNA and protein levels of CELF6 were significantly lower compared to the normal colonic epithelial cell line NCM460 (Fig. 1F,G). These results suggest that CELF6 expression may serve as a diagnostic and prognostic biomarker for CRC.

To investigate the functional role of CELF6 in CRC, we established stable HCT15 and SW48 cell lines with either CELF6 overexpression or knockdown (Fig. 2A). Overexpression of CELF6 resulted in reduced cell viability (Fig. 2B) and decreased colony formation (Fig. 2C,D) in both cell lines. Conversely, CELF6 knockdown led to increased cell proliferation. FCM analysis revealed that CELF6 overexpression induced G1 cell cycle arrest in both HCT15 and SW48 cells, whereas CELF6 knockdown impeded this G1 arrest (Fig. 2E,F). Western blot analysis showed that CELF6 overexpression reduced the protein levels of cell cycle regulators CDK1, cyclin D1, and cyclin B1, while increasing the expression of the cyclin-dependent kinase inhibitor p27^kip1. In contrast, CELF6 knockdown resulted in elevated levels of CDK1, cyclin D1, and cyclin B1, along with decreased levels of p27^kip1 (Fig. 2G). These findings suggest that CELF6 modulates CRC cell proliferation by regulating cell cycle progression.

Fig. 2.

CELF6 inhibits CRC cell proliferation and cell cycle progression. (A) Western blot analysis of CELF6 levels in HCT15 and SW48 cells with CELF6 overexpression or knockdown. CELF6 knockdown was achieved using two different shRNAs (shCELF6-1# and shCELF6-2#). (B) Assessment of cell proliferation by cell counting kit-8 (CCK-8) assays in CELF6 overexpressed or knocked-down HCT15 and SW48 cells. (C,D) Colony formation assays evaluating the impact of CELF6 overexpression or knockdown on cell growth in HCT15 and SW48 cells. (E,F) Flow cytometry analysis of cell cycle distribution in CELF6 overexpressed or knocked-down HCT15 and SW48 cells. (G) Western blot analysis of cell cycle proteins cyclin-dependent kinase 1 (CDK1), cyclin D1, cyclin B1, and p27^kip1 following CELF6 overexpression or knockdown. The results are presented as the mean $\pm$ SD of three independent experiments (n = 3). Statistical significance was determined by comparisons with the normal control (NC) group (*p 0.05; **p 0.01; ***p 0.001) and with the shNC group (^#p 0.05; ^#⁢#p 0.01; ^#⁢#⁢#p 0.001).

To explore the role of CELF6 in regulating stem cell characteristics in CRC, we assessed the expression of CSC markers, including Nanog, OCT4, SOX2, and CD133. Western blot analysis revealed that CELF6 overexpression significantly reduced the levels of these CSC markers, whereas CELF6 knockdown led to increased expression of these markers (Fig. 3A). Sphere formation assays demonstrated that CELF6 overexpression inhibited the sphere-forming ability of HCT15 and SW48 cells, indicating reduced stemness, while CELF6 knockdown enhanced sphere formation (Fig. 3B). Additionally, FCM analysis showed that CELF6 overexpression resulted in a significant reduction in ALDH1 and CD44 activities in HCT15 and SW48 cells. Conversely, CELF6 knockdown increased the activities of ALDH1 and CD44 (Fig. 3C). These results collectively suggest that CELF6 plays a critical role in diminishing the stemness features of CRC cells in vitro.

Fig. 3.

CELF6 suppresses the stemness features of CRC cells. (A) Western blot analysis of stem cell markers Nanog, OCT4, SOX2, and CD133 in HCT15 and SW48 cells with CELF6 overexpression (CELF6-OE) or knockdown (shCELF6-2#). (B) Representative images and quantification of sphere formation assays in HCT15 and SW48 cells with CELF6 overexpression or knockdown. Scale bars, 200 µm. (C) Flow cytometry analysis of ALDH1 and CD44 activities in HCT15 and SW48 cells with CELF6 overexpression or knockdown. The results are presented as the mean $\pm$ SD of three independent experiments (n = 3). Statistical significance was determined by comparisons with the normal control (NC) group (**p 0.01; ***p 0.001) and with the shNC group (^#p 0.05; ^#⁢#p 0.01; ^#⁢#⁢#p 0.001).

To investigate the role of CELF6 in CRC development, we examined its effect on HOXA5 expression. The results indicated that CELF6 positively regulates HOXA5 expression in both HCT15 and SW48 cell lines. Specifically, CELF6 overexpression resulted in a significant increase in HOXA5 expression at both the mRNA and protein levels (Fig. 4A,B). Conversely, CELF6 knockdown led to a decrease in HOXA5 expression. qRT-PCR analysis of CRC tissues confirmed reduced HOXA5 mRNA levels in tumor samples compared to adjacent non-cancerous tissues (Fig. 4C). A positive correlation between CELF6 and HOXA5 mRNA levels was also observed in clinical samples, highlighting a potential regulatory relationship (Fig. 4D). IHC analyses further supported these findings, showing downregulation of HOXA5 protein expression in CRC tissues (Fig. 4E,F). Collectively, these data suggest that HOXA5 is upregulated following CELF6 overexpression and may act as a downstream effector in the regulatory network mediated by CELF6, influencing CRC progression.

Fig. 4.

CELF6 promotes HOXA5 expression in CRC cells. (A,B) Impact of CELF6 overexpression (CELF6-OE) or knockdown (shCELF6-2#) on HOXA5 mRNA and protein levels assessed by qRT-PCR and western blotting. (C) Relative mRNA expression levels of HOXA5 in 60 paired CRC tissues and adjacent non-cancerous tissues measured by qRT-PCR. (D) Correlation analysis between HOXA5 mRNA and CELF6 expression levels in 60 CRC samples. (E,F) Representative IHC images and quantification of HOXA5 protein expression in CRC and adjacent non-cancerous tissues at 100$\times$ and 200$\times$ magnifications. Scale bars, 100µm or 200µm. The results are presented as the mean $\pm$ SD of three independent experiments (n = 3). Statistical significance was determined by comparisons with the normal control (NC) or normal group (**p 0.01; ***p 0.001) and with the shNC group (^#⁢#p 0.01; ^#⁢#⁢#p 0.001).

Regulation of mRNA stability by RNA binding proteins (RBPs) is a well-established mechanism [27]. We hypothesized that CELF6 interacts with HOXA5 and influences its mRNA stability. Actinomycin D (Act D) assays demonstrated a gradual decrease in HOXA5 mRNA levels in control cells, whereas CELF6 overexpression significantly slowed the decay of HOXA5 mRNA (Fig. 5A), indicating that CELF6 stabilizes HOXA5 transcripts.

Fig. 5.

CELF6 positively modulated HOXA5 by binding to its 3^′ UTR. (A) qRT-PCR analysis of HOXA5 mRNA levels in CELF6-overexpressing and control HCT15 and SW48 cells treated with Actinomycin D for the indicated durations. Statistical significance is indicated as *p 0.05; **p 0.01 vs. NC group. (B) Dual luciferase reporter assays showing increased luciferase activity in the presence of HOXA5 3^′ UTR following CELF6 overexpression. Statistical significance is indicated as ***p 0.001 vs. HOXA5-wild-type (WT) group. (C) RNA Immunoprecipitation (RIP) assays detecting interactions between CELF6 and HOXA5 in HCT15 and SW48 cells. Statistical significance is indicated as ***p 0.001 compared to the immunoglobulin G (IgG) group. (D) RNA pull-down assays and western blotting demonstrating CELF6 enrichment in the HOXA5 3^′ UTR in HCT15 and SW48 cells. The results are presented as the mean $\pm$ SD of three independent experiments (n = 3).

To investigate whether CELF6 stabilizes HOXA5 mRNA through direct interaction, we cloned the HOXA5 3^′ UTR and a control vector into luciferase reporter plasmids. These plasmids were introduced into CELF6-overexpressing cell lines and their respective controls. Dual luciferase reporter gene assay showed that in CELF6-overexpressed CRC cells, the luciferase activities of the wild-type HOXA5 3^′ UTR reporter were significantly increased, whereas those of the mutant reporter were not significantly altered (Fig. 5B). Further analysis using RIP assays confirmed a direct interaction between CELF6 protein and HOXA5 mRNA (Fig. 5C). To corroborate this interaction, RNA pull-down assays coupled with western blotting were performed. The results indicated that CELF6 was specifically enriched in the HOXA5 3^′ UTR, with no significant enrichment in the 5^′ UTR or coding region (CR) (Fig. 5D). Overall, these findings suggest that CELF6 mediates the regulation of HOXA5 mRNA stability through direct binding to its 3^′ UTR.

Given that CELF6 regulates HOXA5 mRNA stability and abundance, we aimed to validate HOXA5 as a key mediator of CELF6’s antitumor effects. To investigate this, HCT15 and SW48 cells with CELF6 overexpression were transfected with shRNA targeting HOXA5 or a negative control (shNC). Western blot analysis demonstrated that CELF6 overexpression significantly counteracted the reduction of HOXA5 protein levels caused by HOXA5 knockdown in both cell lines (Fig. 6A).

Fig. 6.

Knockdown of HOXA5 reverses the antitumor activity of CELF6. The HOXA5 gene was knocked down in HCT15 and SW48 cells overexpressing CELF6 through transfection with shHOXA5 or shNC. (A) Western blot analysis of CELF6 and HOXA5 protein levels in HCT15 and SW48 cells overexpressing CELF6 and transfected with shHOXA5 or shRNA. (B,C) Colony formation assays assessing the proliferative potential of CELF6-overexpressing cells with HOXA5 knockdown. (D,E) Flow cytometry analysis of cell cycle distribution in CELF6-overexpressing cells with or without HOXA5 knockdown. (F) Protein levels of Nanog, OCT4, SOX2, and CD133 in CELF6-overexpressing cells with HOXA5 knockdown assessed by western blotting. The results are presented as the mean $\pm$ SD of three independent experiments (n = 3). Statistical significance was determined by comparing with the NC+shNC group (*p 0.05; **p 0.01; ***p 0.001) and with the CELF6-OE+shNC group (^#p 0.05; ^#⁢#p 0.01; ^#⁢#⁢#p 0.001) as well as the shHOXA5+NC group (^{^⁢^⁢^}p 0.001).

Colony formation assays revealed that HOXA5 knockdown significantly restored the proliferative capacity of CELF6-overexpressing cells (Fig. 6B,C). Furthermore, FCM analysis indicated that HOXA5 knockdown reversed the G1 cell cycle arrest induced by CELF6 overexpression (Fig. 6D,E). Additionally, the suppression of HOXA5 attenuated the reduction in stemness markers Nanog, OCT4, SOX2, and CD133 caused by CELF6 overexpression (Fig. 6F). Collectively, these results suggest that HOXA5 plays an essential role in mediating CELF6’s effects on CRC cell aggressiveness.

To investigate the impact of CELF6 on tumorigenesis in vivo, we conducted xenograft experiments using SW48 cells with CELF6 overexpression. Consistent with our in vitro findings, tumors derived from CELF6-overexpressing cells grew significantly slower compared to those from control cells (Fig. 7A). Additionally, the tumor volume and weight were notably reduced in the CELF6 overexpression group (Fig. 7B,C). Additionally, qRT-PCR analysis of xenograft tumors revealed increased mRNA levels of both CELF6 and HOXA5 in the CELF6 overexpression group (Fig. 7D), suggesting that CELF6 modulates HOXA5 expression in vivo. IHC staining of tumor tissue sections further demonstrated elevated expression levels of CELF6 and HOXA5, while the stem cell markers SOX2 and CD133 showed a marked decrease in the CELF6 overexpression group (Fig. 7E,F). These results indicate that CELF6 inhibits CRC tumor growth in vivo through the regulation of HOXA5.

Fig. 7.

CELF6 overexpression ameliorates the CRC cell growth in vivo. Nude mice were inoculated with either CELF6-overexpressing SW48 cells or control cells. (A) Representative images of nude mice and tumor xenografts derived from CELF6-overexpressing and control SW48 cells. (B,C) Measurement of tumor size and weight in CELF6-overexpressing versus control groups. (D) qRT-PCR analysis of CELF6 and HOXA5 expression in xenograft tumors. (E,F) Representative IHC images (scale bar = 200 µm) and statistical analysis of CELF6, HOXA5, SOX2, and CD133 expression in tumor sections. The results are presented as the mean $\pm$ SD of three independent experiments (n = 6). Statistical significance was determined by comparing it with the NC group (***p 0.001).