Bioinformatics analysis

We obtained and filtered the counts file data for CRC patients from The Cancer Genome Atlas (TCGA) database. By leveraging the edgeR package, we conducted an analysis for differentially expressed mRNAs (DEmRNAs) to compare the expression profiles between tumor and normal tissues (log2|FC| > 2, FDR < 0.05). This led to the discovery of 647 tumor tissues and 51 normal tissues, totaling 2185 DEmRNAs, with 1262 upregulated and 923 downregulated. To illuminate the potential role of RBM24 in CRC, we categorized the CRC cohort into high and low expression groups based on the median RBM24 expression and performed gene set enrichment analysis (GSEA) to determine whether genes in these groups were enriched in biological functions of significance.

Cell culture

The human colon epithelial cell line FHC and CRC cell lines HT-29, SW480, and DLD-1 were sourced from the BFB–Shanghai Cell Bank (China). They were cultured in DMEM-H medium (HyClone, USA) with 10% fetal bovine serum (FBS, Invitrogen, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, USA) under conditions of 5% CO₂ at 37°C.

Cell transfection

The RBM24 overexpression (oe-RBM24) and knockdown (si-RBM24) constructs, along with their negative controls (oe-NC, si-NC), were synthesized by Shanghai GeneChem Co., Ltd. The transfection of these vectors into CRC cells was facilitated using Lipofectamine 2000 (Invitrogen, USA) following the provided instructions. Forty-eight hours after transfection, the cells were collected for further experimental procedures.

The CRC cells were incubated with DMSO or PD0325901 (10 µM), an inhibitor of the MAPK pathway, for 48 h, followed by Cell Counting Kit-8 (CCK-8), Western blot (WB), and Transwell [17].

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells using TRIzol reagent, and the RNA concentration and purity were assessed with a NanoDrop One UV-Vis spectrophotometer. The reverse transcription of RNA to cDNA was facilitated by SuperScript III Reverse Transcriptase (Invitrogen, USA). The qRT-PCR was performed on the ABI 7500 PCR system (Applied Biosystems, USA) with the Platinum SYBR Green qPCR SuperMix UDG kit (Invitrogen, USA). The relative expression levels of RBM24 were determined using the 2^–ΔΔCT method, with GAPDH serving as the internal control. The primer sequences are detailed in the Table I provided.

WB

Cells were collected and then lysed in RIPA lysis buffer (Beyotime, China) containing 1% protease and phosphatase inhibitors (Beyotime, China) at 4°C to extract total proteins. The protein concentration was quantified using the bicinchoninic acid (BCA) assay kit (Beyotime, China). The BCA protein assay kit (Beyotime, China) was employed to measure protein concentrations. Denatured proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 2 h at 37°C and incubated overnight at 4°C with primary antibodies for MMP2 (abcam, UK), MMP9 (abcam, UK), E-cadherin (CST, USA), fibronectin (CST, USA), p-ERK/ERK (abcam, UK), p-JNK/JNK (abcam, UK), FDX1 (Abclonal, China), DLAT (Abclonal, China), LIAS (abcam, UK), RBM24 (Proteintech Group, USA), and GAPDH (abcam, UK). The membranes were further incubated with HRP-conjugated secondary goat anti-rabbit IgG (abcam, UK) for 2 h at room temperature. Chemiluminescent detection was performed using the ChemiScope 6000 system (Clinx, China) with NcmECL Ultra (NCM Biotech, China) for imaging.

CCK-8 assay

Cell proliferation was determined by the CCK-8 kit (Dojindo, Japan). CRC cells were dispensed into a 96-well plate at a concentration of 2 × 10³ cells per well. After cell adherence, 10 µl of CCK-8 reagent was introduced at 0 h, 24 h, 48 h, and 72 h. The plate was incubated in a cell culture incubator away from light for 2 h, and the absorbance at 450 nm in each well was measured with a microplate reader.

Transwell assay

CRC cells, 48 h after transfection, were resuspended in serum-free medium and placed into the upper compartment of the Transwell at a density of 2 × 10⁴ cells per well with 200 µl of the medium. The lower compartment received 600 µl of medium supplemented with 10% FBS. After a 24-h incubation, cells that had migrated to the exterior of the Transwell membrane were fixed using 4% paraformaldehyde and stained with 0.1% crystal violet. For the invasion assay, cells were seeded in the upper chamber coated with Matrigel, following the same protocol as the migration assay. The number of cells that migrated or invaded was counted under a microscope by selecting three random fields and calculating the mean cell count.

Immunofluorescence (IF)

The cells were fixed in 75% alcohol for 30 min. Subsequently, they were treated with 0.1% Triton X-100 for permeabilization for 10 min. Next, they were blocked with 5% BSA for 1 h. RBM24 antibody (Proteintech Group, USA) was applied, followed by incubation with a Cy3-conjugated secondary antibody (Abclonal, China). After being washed with PBS, they were observed using an inverted fluorescence microscope.

Measurement of intracellular copper content

HT-29 cells were cultured in a 6-well plate overnight. On the following day, 200 nM elesclomol-Cu (Es-Cu) was added to the cells, obtained from MCE (USA), at a 1 : 1 ratio. After incubation for 24 h, the cells were gathered and resuspended in 100 µl of PBS. The Copper (Cu) Content Assay Kit (Solarbio, China) was used to quantify the copper ion concentration inside the cells, according to the supplier’s guidelines [18].

Data analysis

Experiments were conducted in triplicate, and data were analyzed using GraphPad Prism 8 software. The results are reported as mean ± SD. Student’s t-test was chosen for contrasting pairs of groups, whereas the one-way ANOVA was the protocol for assessing differences across multiple groups. P < 0.05 was deemed statistically significant.

Overexpression of RBM24 inhibited CRC metastasis

As part of our investigation to identify mRNAs involved in the development of CRC, bioinformatics analysis showed that RBM24 expression was downregulated in CRC tissues (Figure 1 A). qPCR and IF experiments demonstrated significant downregulation of RBM24 expression in CRC cell lines HT-29, SW480, and DLD-1 when compared to the FHC human colon epithelial cell line (Figures 1 B, C). Furthermore, the establishment of oe-NC/oe-RBM24 groups within HT-29 and SW480 cells was executed. qRT-PCR analyses confirmed a significant increase in RBM24 expression in the oe-RBM24 group compared with the oe-NC group (Figure 1 D). The CCK-8 and Transwell assay data showed that the overexpression of RBM24 hindered the proliferative, migratory, and invasive capacities of CRC cells (Figures 1 E, F). Following WB analysis of genes related to migration and invasion (MMP2, MMP9) as well as EMT markers (E-cadherin, fibronectin), the data revealed a marked reduction in the expression levels of MMP2, MMP9, and fibronectin, alongside elevated E-cadherin expression in HT-29 and SW480 cells overexpressing RBM24 (Figure 1 G). In conclusion, the outcomes of this investigation strongly suggest downregulation of RBM24 expression in CRC. Moreover, the overexpression of RBM24 was found to inhibit the metastatic capabilities of CRC cells.

Figure 1

Downregulation of RBM24 in CRC suppresses tumor metastasis. A – Bioinformatics analysis of RBM24 expression levels; B, C – qRT-PCR and IF detection of RBM24 expression in CRC cell lines and colonic epithelial cell lines. D – qRT-PCR confirmation of RBM24 overexpression in HT-29 and SW480 cells; E – CCK-8 assay for cell proliferation assessment; F – Transwell migration and invasion assays for HT-29 cells. G – WB detection of migration and invasion-related (MMP2, MMP9) and EMT-related (E-cadherin, fibronectin) gene expression. All experiments were independently repeated three times, and the mean ± standard deviation was calculated. *P < 0.05

RBM24 inhibited the metastatic potential of CRC via the MAPK signaling cascade

To investigate the mechanisms of RBM24 in controlling the migration of CRC cells, we conducted GSEA analysis and found that RBM24 was associated with the MAPK signaling pathway (Figure 2 A). The MAPK pathway is recognized in cellular function, and its aberrant activation can facilitate the EMT process [19]. Subsequently, we silenced RBM24 and further treated HT-29 cells with the MAPK pathway inhibitor PD0325901. The qRT-PCR results showed that the RBM24 expression was suppressed in both the si-RBM24 + DMSO and si-RBM24 + PD0325901 groups compared to the si-NC + DMSO group (Figure 2 B). WB results revealed that in HT-29 cells with low RBM24 expression, levels of p-ERK and p-JNK were elevated, but no significant change was observed in the levels of ERK and JNK. The application of PD0325901 impaired the stimulatory effect of low RBM24 expression on the MAPK signaling pathway (Figures 2 C, D). We also evaluated the proliferative, migratory, and invasive capabilities of HT-29 cells using CCK-8 and Transwell assays. It was found that low RBM24 expression promoted the proliferation, migration, and invasion of HT-29 cells, and the addition of PD0325901 impaired the influence of low RBM24 expression on the biological behavior of CRC cells (Figures 2 E, F). Another WB analysis revealed that the knockdown of RBM24 led to upregulation of MMP2, MMP9, and fibronectin expression, and a downregulation of E-cadherin expression, and the addition of PD0325901 returned the protein expression levels to control levels (Figure 2 G). Overall, inhibition of the MAPK signaling pathway counteracted the metastatic-promoting effect of low RBM24 expression in CRC.

Figure 2

RBM24 impeded CRC metastatic spread by modulating the MAPK pathway. A – The interaction of RBM24 with the MAPK signaling pathway (NES = 1.55, FDR = 0.347); B – qRT-PCR measurement of RBM24 expression in groups receiving si- NC + DMSO, si-RBM24 + DMSO, and si-RBM24 + PD0325901; C, D – WB evaluation of the expression of proteins in the MAPK signaling pathway (p-ERK/ERK, p-JNK/JNK); E – CCK-8 assessment of cell proliferation in different groups; F – Transwell assays evaluated HT-29 cell migration and invasion; G – WB analysis of E-cadherin, fibronectin, MMP2, and MMP9 expression levels. All experiments were independently repeated three times, and the mean ± standard deviation was calculated. *P < 0.05 when compared to si-NC + DMSO; #p < 0.05 when compared to si-RBM24+DMSO; ns denotes p > 0.05 when compared to si-NC + DMSO

Cuproptosis upregulated RBM24 expression in CRC cells

To probe into the molecular mechanisms that govern RBM24 expression, we performed bioinformatics analysis, where a connection between RBM24 and copper ion homeostasis pathways was identified (Figure 3 A). Subsequently, we detected the expression of cuproptosis-related proteins in the oe-RBM24/oe-NC group HT-29 cells. The WB experimental results showed that after overexpression of RBM24, the expression levels of FDX1, DLAT, and LIAS were significantly downregulated, and cuproptosis was activated (Figure 3 B). Research has highlighted the pivotal role of cuproptosis in tumor metastasis [20]. Therefore, we hypothesized that the expression of RBM24 might be related to the occurrence of cuproptosis. To test this, we treated HT-29 cells with Es-Cu and measured much higher intracellular copper ion concentrations than in the NC group (Figure 3 C). Additionally, CCK-8 assay results indicated significant inhibition of CRC cell proliferation with Es-Cu treatment (Figure 3 D). The qRT-PCR results supported that Es-Cu treatment significantly increased RBM24 expression (Figure 3 E). Finally, we detected the protein expression levels of cuproptosis-related genes and RBM24 through WB. The results showed that after treatment with Es-Cu, the expression levels of FDX1, DLAT, and LIAS were significantly downregulated, while the expression of RBM24 was significantly upregulated (Figure 3 F). These results collectively indicated that the treatment of CRC cells with copper ionophores and copper ions could upregulate the expression of RBM24.

Figure 3

Cuproptosis upregulated RBM24 expression in CRC cells. A – Enrichment of RBM24 in pathways associated with copper ion homeostasis (NES = 0.81, FDR = 0.822); B – WB detection of cuproptosis- related proteins (FDX1, DLAT, and LIAS); C – HT-29 cells treated with 200 nM Es-Cu (1 : 1 ratio), followed by determination of copper ion level using a detection kit; D – CCK-8 assay to evaluate cell proliferation in both groups; E – qRT-PCR to measure RBM24 mRNA expression levels in both groups; F – WB detection of FDX1, DLAT, LIAS and RBM24 in both groups. All experiments were independently repeated three times, and the mean ± standard deviation was calculated. *P < 0.05

Cuproptosis inhibited metastasis of CRC cells through the RBM24/MAPK axis

To determine whether cuproptosis influences CRC metastasis through the upregulation of RBM24, Es-Cu was introduced to CRC cells with suppressed RBM24 expression, followed by the assessment of RBM24 expression via qRT-PCR. It was observed that the Es-Cu + si-NC group exhibited markedly increased RBM24 expression compared to the DMSO + si-NC group, whereas the Es-Cu + si-RBM24 group exhibited attenuation of the upregulating effect of Es-Cu on RBM24 expression (Figure 4 A). Thereafter, we examined the expression of p-ERK/ERK and p-JNK/JNK by WB and discovered that Es-Cu treatment inhibited the expression of the phosphorylated forms, with the reduction in MAPK signaling pathway gene expression being relieved after RBM24 knockdown (Figure 4 B). In addition, CCK-8 and Transwell assay results showed that after Es-Cu treatment, the proliferation, migration, and invasion abilities of HT-29 cells significantly decreased, while knockdown of RBM24 concurrently led to increased recovery (Figures 4 C–E). WB also demonstrated that Es-Cu treatment downregulated the expression of MMP2, MMP9, and fibronectin, and upregulated E-cadherin expression. Knockdown of RBM24 attenuated the changes in migration, invasion, and EMT-related protein expression levels induced by Es-Cu treatment (Figures 4 F, G). We concluded that elevated levels of cuproptosis disrupted the MAPK signaling cascade, thereby inhibiting CRC cell spread.

Figure 4

Cuproptosis inhibits the metastasis of CRC cells through the RBM24/MAPK axis. A – qRT-PCR determination of RBM24 levels in the DMSO + si-NC, Es-Cu + si-NC, and Es-Cu + si-RBM24 conditions; B – WB analysis quantified the expression of p-ERK/ERK and p-JNK/JNK; C – CCK-8 assay assessing the proliferation rate of HT-29 cells across the groups; D, E – Transwell migration and invasion assays for HT-29 cells; F, G – WB analysis for expression of E-cadherin, fibronectin, and MMP2, MMP9. All experiments were independently repeated three times, and the mean ± standard deviation was calculated. *P < 0.05 compared to the DMSO + si-NC group; *P < 0.05 compared to the DMSO + si-NC group; #p < 0.05 compared to the Es-Cu + si-NC group; ns denotes p > 0.05 when compared to si-NC + DMSO