CASC19 stabilization by ALYREF via m5C modification and its impact on SCD in colorectal cancer cell aggressiveness and stemness.

Introduction
Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and poses a serious threat to human health [1, 2]. According to the GLOBOCAN 2022 global cancer statistics, CRC ranks among the top three malignancies in terms of incidence and mortality [3]. In recent years, its incidence has been rising among younger populations [4, 5]. The development of CRC is typically accompanied by complex genetic and epigenetic alterations, involving multiple dysregulated signaling pathways [6]. Although advancements in surgery, radiotherapy, chemotherapy, and targeted therapies have improved the prognosis of some CRC patients, effective treatment options remain limited for those with advanced or recurrent CRC, and challenges such as tumor resistance and recurrence persist [7–9]. Therefore, an in-depth exploration of the molecular mechanisms underlying CRC pathogenesis, as well as the identification of novel molecular biomarkers and potential therapeutic targets, is crucial for improving early diagnosis and patient prognosis.
Aly/REF export factor (ALYREF) is an mRNA export factor that plays a key role in the nuclear export of mRNA [10, 11]. Additionally, ALYREF is an important regulator of RNA modifications, including N6-methyladenosine and 5-methylcytosine (m5C) [12, 13]. Studies have shown that ALYREF facilitates mRNA nuclear export by binding to m5C modification sites, thereby regulating gene stability and expression levels [13, 14]. In various malignancies, such as hepatocellular carcinoma [15], breast cancer [16], and lung cancer [17], ALYREF has been found to be closely associated with tumor progression and has been proposed as a potential cancer biomarker. However, its role in CRC remains largely unexplored. A previous study reported that ALYREF is highly expressed in CRC tissues and is associated with tumorigenesis and progression [18]. Nevertheless, the precise molecular mechanisms of ALYREF in CRC and its correlation with clinicopathological features have not been systematically investigated. Given the critical role of ALYREF in RNA modification and stability regulation, we hypothesize that it may influence CRC progression by epigenetically regulating downstream long non-coding RNAs (lncRNAs) or protein-coding genes. Therefore, a comprehensive investigation of ALYREF’s function and its underlying molecular mechanisms in CRC will enhance our understanding of CRC pathogenesis and may provide new insights into early diagnosis and personalized treatment strategies.
This study aims to elucidate how ALYREF regulates lncRNA cancer susceptibility 19 (CASC19) stability through m5C modification and further investigate how CASC19 recruits heterogeneous nuclear ribonucleoprotein C (HNRNPC) to stabilize stearoyl-CoA desaturase (SCD), thereby promoting CRC cell proliferation, migration, invasion, and stemness. Our findings will reveal the crucial role of ALYREF in CRC and its regulatory network, providing new theoretical foundations and potential therapeutic targets for precision diagnosis and treatment of CRC.

Materials and methods

Bioinformatics analysis
Gene expression datasets related to CRC were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases for bioinformatics analysis. TCGA data were accessed via the GEPIA platform, while GEO datasets were retrieved using the GEOquery R package (v2.60.0). Survival analysis was performed with the survival (v3.5.7) and survminer (v0.4.9) packages to evaluate correlations between ALYREF, CASC19, HNRNPC, and SCD expression levels and CRC patient prognosis.

Patients and tissue samples
A cohort of 70 treatment-naïve CRC patients was enrolled at Affiliated Nanhua Hospital between June 2022 and June 2023. Tumor tissues and paired adjacent normal tissues were surgically resected, histologically confirmed, rapidly frozen in liquid nitrogen, and stored at -80 °C until analysis. Written informed consent was obtained from all participants, and the study protocol was approved by the Ethics Committee of the Affiliated Nanhua Hospital (Approval No. 2025-KY-034).

Cell culture
Human CRC cell lines (LOVO, RKO, SW480, SW620, DLD1, HT-29, HCT116) and normal colorectal epithelial cells (FHC, ATCC) were maintained in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Cat. No. 11875-093) supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, Cat. No. FSP500) and 1% penicillin-streptomycin (Biosharp, Cat. No. BL505A) at 37 °C under 5% CO₂. Cells were passaged using 0.25% trypsin-EDTA (Gibco, Cat. No. 25200-056) and cryopreserved in FBS-based freezing medium containing 10% DMSO (Beyotime, Cat. No.ST038-100 ml).

Lentiviral ShRNA production and infection
ALYREF-targeting shRNA sequences (shRNA-1: 5′-GGAAACTGCTGGTGTCCAATC-3′; shRNA-2: 5′-GAATTCAAAGCAGCAGCTTTC-3′) were cloned into the pLKO.1 vector (GenePharma). HEK293T cells (ATCC) were co-transfected with shRNA plasmids and packaging plasmids (psPAX2/pMD2.G, Addgene) using Lipofectamine 3000 (Invitrogen, Cat. No. L3000015). Viral supernatants harvested 48 h post-transfection were filtered (0.45 μm) and used to infect CRC cells with 8 µg/mL polybrene (Sigma, Cat. No. TR-1003-G). Stable knockdown lines were selected with 2 µg/mL puromycin (Sigma, Cat. No. P8833).

Small interfering RNA (siRNA) treatment
siRNAs targeting NSUN2, CASC19, HNRNPC, and SCD (GenePharma) were designed using NCBI RefSeq and validated via BLAST to minimize off-target effects. Cells at 50–60% confluence in 6-well plates were transfected with 50 nM siRNA using Lipofectamine RNAiMAX (Invitrogen, Cat. No. 13778150). Knockdown efficiency was verified by qPCR and Western blot after 48 h, as previously described.

Overexpression plasmid construction and transfection
Full-length ALYREF, NSUN2, CASC19, and HNRNPC cDNAs (GenePharma) were cloned into the pcDNA3.1 vector (Invitrogen). Transfections were performed in 6-well plates at 60–70% cell density using Lipofectamine 3000 (2.5 µg plasmid/well) (Invitrogen, Cat. No. L3000015). Empty vector served as the negative control. Overexpression efficiency was confirmed via qPCR and Western blot at 48 h prior to functional assays.

Cell migration and invasion assays
Cell migration assays were conducted using serum-free Transwell chambers (8-µm pore size, Corning, USA). A total of 5 × 10⁴ cells were seeded in each upper chamber with serum-free medium, while the lower chamber was supplemented with 10% FBS-containing medium to provide a chemotactic gradient. After 24 h, the chambers were washed with PBS, fixed in 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 30 min. Non-migrated cells were removed, and migrated cells were counted in five randomly selected microscopic fields. The invasion assay followed the same procedure as the migration assay, except that the upper surface of the Transwell chamber was pre-coated with 50 µL Matrigel (BD Biosciences, USA). All experiments were repeated three times to ensure data reliability.

Tumorsphere formation assay
Single-cell suspensions (4 × 10⁴ cells/well) were cultured in ultra-low attachment plates (Corning) with serum-free DMEM/F-12 medium (Invitrogen, Cat. No. 11320-033) containing 10 ng/mL B-FGF (CST, Cat. No. 46879), 20 ng/mL EGF (CST, Cat. No. 8916), 20 ng/mL IGF (CST, Cat. No. 29608), and B27 supplement (Gibco, Cat. No. 17504-044). Tumorspheres were imaged after 10–14 days, counted microscopically, and sized using ImageJ (50–100 μm, 100–150 μm, > 150 μm).

Cell counting kit-8 (CCK-8) assay
Cells (1 × 10⁴/well) in 96-well plates were treated with CCK-8 reagent (Beyotime, Cat. No. C0037) at 0, 24, 48, and 72 h. Absorbance at 490 nm was measured after 4 h incubation (Thermo Fisher microplate reader). Triplicate experiments generated proliferation curves.

Flow cytometry for cell cycle and apoptosis analysis
Cell cycle analysis: Propidium iodide (PI) staining was used to analyze the cell cycle. Cells were collected and fixed with pre-cooled 70% ethanol at -20 °C overnight. After washing with PBS, the cells were incubated with 50 µg/mL RNase A (Sigma-Aldrich, Cat. No. R4875) at 37 °C for 30 min, followed by 50 µg/mL PI staining (Sigma-Aldrich, Cat. No. P4170) for 15 min. The cell cycle distribution was detected using a flow cytometer (BD FACSCalibur, USA) and analyzed with FlowJo software (version 10.8.1).
Apoptosis analysis: Annexin V-FITC/PI double staining was performed. After washing with PBS, cells were resuspended in 1× Annexin V binding buffer (BD Biosciences, USA). 5 µL Annexin V-FITC (BD Biosciences, Cat. No. 556547) and 5 µL PI were added, and the samples were incubated in the dark for 15 min. Apoptosis rates were measured by flow cytometry, and the proportions of early and late apoptotic cells were analyzed using FlowJo software (version 10.8.1).

Quantitative real-time PCR (qPCR)
Total RNA extracted with TRIzol (Invitrogen, Cat. No. 15596026) was reverse-transcribed using a High-Capacity cDNA Kit (Thermo Fisher, Cat. No. 4368814). SYBR Green Master Mix (Applied Biosystems, Cat. No. A25742) and StepOnePlus system amplified target genes, with GAPDH as the endogenous control. Relative expression was calculated via the 2⁻ΔΔCT method. All primer sequences are listed in Table 1, and each experiment was performed in triplicate to ensure data reliability.

Western blot
Cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Sigma Chemicals, UK) to prevent protein degradation. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, Cat. No. P0009). Equal amounts of protein were separated via SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin at room temperature for 1 h to reduce non-specific binding. Subsequently, membranes were incubated overnight at 4 °C with the following primary antibodies: ALDHA1 (ab52492, 1:1000), CD133 (ab222782, 1:5000), CD44 (ab51037, 1:1000), SOX2 (ab92494, 1:1000), HNRNPC (ab133607, 1:10000), SCD (ab181845, 1:1000), GAPDH (ab8245, 1:5000) (All antibodies were purchased from Abcam, USA). The next day, membranes were washed with PBS and incubated with horseradish peroxidase -conjugated secondary antibodies (Santa Cruz, USA) at 37 °C for 1 h. Protein bands were detected using enhanced chemiluminescence and analyzed using ImageJ software. GAPDH was used as an internal control for normalization.

RNA pull-down and Truncation assay
Cells were lysed using lysis buffer and subjected to ultrasonic fragmentation at 4 °C. The cell lysate was incubated with biotin-labeled RNA probes pre-bound to streptavidin magnetic beads, followed by rotation at 4 °C for 3 h. The beads were then washed to remove nonspecific bindings. The RNA-protein complexes captured on the beads were digested with proteinase K, and the RNA-binding proteins were analyzed by Western blot to validate the interaction between target proteins and RNA. Additionally, truncation assays were performed using deletion or mutant RNA probes to further determine the specific RNA binding sites essential for protein interaction.

RNA Immunoprecipitation (RIP)
The RIP assay was performed using a commercial RIP kit (Genseed, China, Cat. No. GS-RIP-01). Approximately 1 × 10⁷ cells were collected and lysed with RIP lysis buffer (Beyotime, Cat. No. P1005). Antibodies against ALYREF (Cat. No. ab6141), SCD (Cat. No. ab10294), HNRNPC (Cat. No. ab19862), and m5C (Cat. No. ab10805) (Abcam), or an IgG isotype control (5 µg, Abcam, Cat. No. ab172730), were pre-incubated with magnetic beads at 4 °C for 2 h. The cell lysate was then added and incubated overnight at 4 °C under rotation. After washing, the co-precipitated RNA was extracted and analyzed by qPCR to assess the enrichment of CASC19, SCD, and other target RNAs, evaluating their interactions with specific proteins.

RNA stability assay
Cells were treated with 5 µg/mL actinomycin D (Sigma Aldrich, USA, Cat. No. A1410) for 0, 2, 4, and 6 h. Total RNA was extracted at each time point, and the relative expression levels of CASC19 and SCD were determined by qRT-PCR. GAPDH was used as an internal control for normalization. The RNA half-life was calculated to evaluate changes in the stability of CASC19 and SCD mRNA.

Methylated RNA Immunoprecipitation (MeRIP)
The m5C modification level of CASC19 was detected using the Magna MeRIP m5C kit (Merck, Germany, Cat. No. 17-10499). Total RNA was extracted with TRIzol reagent (Invitrogen) and treated with DNase to remove genomic DNA contamination. RNA samples were incubated with either an anti-m5C antibody or an IgG control at 4 °C overnight under rotation. Protein A/G magnetic beads were then added, and incubation continued at 4 °C. The bound RNA was eluted and analyzed by qPCR to assess CASC19 enrichment and its m5C modification levels.

In vivo xenograft model
All animal experiments were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the institutional animal ethics committee. Four-week-old male NOD-SCID mice were maintained under specific pathogen-free conditions.
For the subcutaneous tumorigenesis patient-derived xenograft (PDX) model, freshly resected colorectal cancer tissues from patients were cut into small fragments (approximately 3 × 3 × 3 mm³) and subcutaneously implanted into the flanks of NOD-SCID mice under anesthesia. Tumor volume was measured every three days using a caliper, and the growth curve was plotted according to the formula: volume = 0.5 × length × width². When the tumor volume reached approximately 50 mm³, mice were injected intratumorally with lentivirus carrying the indicated constructs. At the experimental endpoint, tumor xenografts were excised for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis.
For the lung metastasis model, RKO or HCT116 cells (5 × 10⁶ cells per mouse) were suspended in 200 µL phosphate-buffered saline (PBS) and injected into the tail vein of NOD-SCID mice (n = 5 per group). After 6–8 weeks, the mice were euthanized, and lung tissues were harvested, fixed, and subjected to H&E staining and IHC analysis to evaluate metastatic nodules.

IHC staining
Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. After deparaffinization with xylene, rehydration through a graded ethanol series, antigen retrieval with sodium citrate buffer, and blocking of endogenous peroxidase activity with 3% H₂O₂, the sections were incubated with primary antibodies against ALYREF (ab6141, 1:500, Abcam), SOX2 (#3728, 1:100, CST, USA), Ki67 (#9449, 1:100, CST), and Bcl-2 (#15071, 1:100, CST) at 4 °C overnight. The next day, sections were incubated with HRP-conjugated biotinylated goat anti-rabbit secondary antibody (ab205718,1:3000, Abcam) at 37 °C for 1 h. Color development was performed using a DAB HRP kit (Beyotime, P0203), followed by counterstaining with hematoxylin (Sigma Aldrich). After dehydration, clearing, and mounting, tissue sections were observed and imaged under a microscope.

H&E staining
After fixation, tissue samples were embedded in paraffin and sectioned at 4 μm thickness. Sections were deparaffinized with xylene, rehydrated through a graded ethanol series, and stained with hematoxylin for 5 min, followed by thorough rinsing with running water. Sections were then differentiated with 0.5% hydrochloric acid ethanol, slightly blued, and stained with eosin for 3 min. After dehydration through a graded ethanol series and clearing with xylene, sections were mounted and observed under a microscope for histological evaluation.

Statistical analysis
All data were analyzed using GraphPad Prism 9.5.1 (GraphPad Software, USA) and SPSS Statistics 26.0 (IBM, USA). Continuous variables are presented as mean ± standard deviation (SD) from at least three independent experiments. Comparisons between two groups were performed using unpaired two-tailed Student’s t-test if the data were normally distributed, or Mann-Whitney U test for non-parametric data. For comparisons among multiple groups, one-way or two-way ANOVA followed by Tukey’s post hoc test was applied. Correlations between variables were assessed using Pearson correlation for normally distributed data and Spearman rank correlation for non-normally distributed data. Kaplan-Meier survival curves were generated to evaluate patient survival, and differences between groups were analyzed using the log-rank test. A two-sided P value < 0.05 was considered statistically significant. Graphs were plotted using GraphPad Prism. All statistical tests and assumptions were clearly checked for normality and variance homogeneity to ensure validity and reproducibility.

Results

ALYREF is highly expressed in clinical CRC samples and is associated with poor prognosis
Analysis of m5C-related genes in the GSE211831 dataset identified significant upregulation of NOP2, NSUN2, ALYREF, and YBX1 in CRC tissues, with ALYREF showing the most pronounced elevation (Fig. 1A). Positive correlations were observed between ALYREF and YBX1, NOP2, and NSUN2 (Fig. 1B), implicating its central role in m5C regulation. Validation using TCGA-COAD/READ cohorts confirmed elevated ALYREF expression in both colon and rectal adenocarcinomas versus normal tissues (Fig. 1C). IHC of clinical specimens further corroborated ALYREF protein overexpression in tumors relative to adjacent non-cancerous tissues (Fig. 1D).

Stratified analysis of 70 CRC patients revealed inverse correlations between ALYREF expression and advanced tumor size (Fig. 1E), T stage (Fig. 1F), and TNM stage (Fig. 1G), while no significant associations were observed with age, sex, tumor location, or differentiation status (Fig. 1H–L). Kaplan-Meier survival analysis demonstrated markedly reduced overall survival in patients with high ALYREF expression (Fig. 1N), supporting its prognostic relevance.
Consistent with bioinformatics findings, qPCR and Western blot analyses of paired tumor/adjacent tissues confirmed significant upregulation of ALYREF mRNA and protein in CRC specimens (Fig. 1O–P). Comparative evaluation across CRC cell lines (LOVO, RKO, SW480, SW620, DLD1, HT-29, HCT116) versus normal FHC cells revealed highest ALYREF expression in HT-29 and HCT116 lines, contrasting with minimal expression in FHC, LOVO, and RKO (Fig. 1Q–R). Based on this differential expression profile, RKO (low ALYREF) and HCT116 (high ALYREF) were selected for functional studies.

ALYREF promotes CRC cell proliferation, colony formation, cell cycle progression, inhibits apoptosis, and enhances migration, invasion, and stemness
Previous studies have confirmed that ALYREF is highly expressed in CRC tissues and is associated with poor prognosis [18]. However, whether ALYREF plays a functional role in CRC cell behavior remains unclear. To further explore the biological functions of ALYREF, we established an ALYREF overexpression system (ALYREF-OE) in RKO cells and an ALYREF knockdown system (sh-ALYREF-1 and sh-ALYREF-2) in HCT116 cells (Fig. 2A, B). We then systematically evaluated its effects on CRC cell proliferation, apoptosis, migration, invasion, and stemness. The results showed that ALYREF overexpression significantly enhanced the proliferative capacity of RKO cells, whereas ALYREF knockdown suppressed cell viability in HCT116 cells (Fig. 2C). Colony formation assays further demonstrated that ALYREF overexpression markedly increased the colony-forming ability of RKO cells, while ALYREF knockdown significantly reduced the number of colonies formed by HCT116 cells (Fig. 2D), reflecting enhanced long-term survival and clonogenic potential. Notably, colonies formed in ALYREF-overexpressing cells were also larger in size, suggesting increased proliferative capacity at the single-cell level. Collectively, these findings indicate that ALYREF promotes CRC progression by enhancing cell viability, clonogenic potential, and proliferative capacity. Migration and invasion assays revealed that ALYREF overexpression significantly enhanced the migratory (Fig. 2E) and invasive abilities (Fig. 2E) of RKO cells, whereas ALYREF knockdown inhibited the migration and invasion of HCT116 cells (Fig. 2E). These results indicate that ALYREF may contribute to CRC metastasis.

Furthermore, ALYREF overexpression decreased the apoptosis rate of RKO cells (Fig. 2F), while ALYREF knockdown significantly increased apoptosis in HCT116 cells (Fig. 2F). Additionally, cell cycle analysis revealed that ALYREF overexpression accelerated cell cycle progression in RKO cells, leading to an increased proportion of S-phase cells (Fig. 2G). In contrast, ALYREF knockdown induced G1-phase arrest in HCT116 cells, suggesting that ALYREF may promote cell proliferation by inhibiting apoptosis and accelerating cell cycle progression. Tumorsphere formation assays demonstrated that ALYREF overexpression significantly enhanced the sphere-forming ability of RKO cells, whereas ALYREF knockdown suppressed tumorsphere formation in HCT116 cells (Fig. 2H). Moreover, the expression levels of cancer stem cell markers [19, 20], including CD133, CD44, SOX2, and ALDHA1, were significantly upregulated in ALYREF-overexpressing RKO cells (Fig. 2I), while these markers were downregulated in ALYREF-knockdown HCT116 cells (Fig. 2I), supporting a critical role for ALYREF in maintaining CRC stemness. In summary, ALYREF may serve as a key driver of CRC progression, providing theoretical evidence for its potential as a therapeutic target.

ALYREF promotes growth and metastasis of PDX CRC
To investigate the oncogenic role of ALYREF in CRC, we established PDX models in NOD/SCID mice to evaluate the effects of ALYREF on tumor growth and metastasis. First, we subcutaneously injected ALYREF-overexpressing (ALYREF-OE) RKO cells and ALYREF-knockdown (sh-ALYREF) HCT116 cells into NOD/SCID mice (Fig. 3A), successfully generating CRC xenograft tumors (Fig. 3B, C). Regular tumor monitoring revealed that compared to the control group, the ALYREF-OE group exhibited significantly increased tumor weight (Fig. 3D) and tumor volume (Fig. 3E), whereas the sh-ALYREF group showed a significant reduction in both tumor weight and volume (Fig. 3D, E). H&E staining showed that tumors in the ALYREF-OE group had higher cellular density, tightly packed cell arrangements, and increased mitotic activity (Fig. 3F), whereas the sh-ALYREF group displayed looser cell arrangements and reduced mitotic activity (Fig. 3F), suggesting that ALYREF promotes CRC cell proliferation and sustains tumor cell viability. IHC analysis further demonstrated that ALYREF-overexpressing tumors had markedly higher expression levels of ALYREF (Fig. 3F and G), SOX2 (Fig. 3F and H), and Ki67 (Fig. 3F and I), whereas Bcl-2 expression was relatively low (Fig. 3F and J). These findings suggest that ALYREF accelerates tumor progression by promoting cell proliferation (Ki67) and maintaining stemness properties (SOX2), while potentially affecting apoptotic pathways by suppressing Bcl-2 expression. Conversely, the sh-ALYREF group exhibited opposite trends, further supporting the oncogenic role of ALYREF in vivo.

To assess the metastatic potential of ALYREF, in vivo imaging and fluorescence signal detection were performed (Fig. 4A). The results showed that the ALYREF-OE group had significantly enhanced pulmonary fluorescence signals, indicating an increased metastatic burden, whereas the sh-ALYREF group displayed markedly reduced fluorescence intensity, suggesting decreased lung metastasis. Gross examination of lung tissues further confirmed these findings, with a significantly higher number of metastatic lung nodules in the ALYREF-OE group and a markedly lower number in the sh-ALYREF group (Fig. 4B). To further verify lung metastasis, we performed H&E staining on lung tissues, which revealed a greater number of metastatic lesions and more extensive cancer cell infiltration in the ALYREF-OE group, whereas the sh-ALYREF group exhibited significantly fewer metastatic foci (Fig. 4C). These results indicate that ALYREF not only enhances CRC tumor growth but also promotes distant organ metastasis, accelerating disease progression. This further supports the potential of ALYREF as a therapeutic target for CRC.

ALYREF stabilizes CASC19 expression via m5C modification
The oncogenic mechanism of ALYREF piqued our interest. Through multi-database analysis, including m5C_RM2Target, ENCORI, POSTAR3, and the GSE211831 dataset (logFC > 4), we identified CASC19 as one of the downstream targets of ALYREF (Fig. 5A). This finding suggests that ALYREF may exert its function in CRC by regulating CASC19 expression. Notably, previous study also demonstrated that high CASC19 expression is associated with malignant clinical phenotypes and poor prognosis in CRC [21, 22]. Therefore, we sought to investigate the relationship between ALYREF and CASC19 and further elucidate its underlying mechanism. Analysis of the TCGA-COAD and TCGA-READ datasets revealed that CASC19 expression was significantly upregulated in both datasets, exhibiting a pattern consistent with ALYREF expression (Fig. 5B). Furthermore, in ALYREF-overexpressing RKO cells and ALYREF-knockdown HCT116 cells, CASC19 expression fluctuated in response to ALYREF modulation (Fig. 5C), further confirming their correlation.

To explore the interaction between ALYREF and CASC19, we conducted RIP and RNA pull-down assays, which demonstrated a significant binding interaction between CASC19 and ALYREF (Fig. 5D and E). Next, to investigate whether ALYREF influences CASC19 stability, we performed m5C-MeRIP assays, which confirmed the presence of m5C modifications on CASC19 (Fig. 5F). This modification is crucial for RNA stability and function. Further experiments using CASC19 mutant constructs revealed that only wild-type (WT) CASC19 was capable of binding to ALYREF, while the mutant version failed to do so (Fig. 5G). Additionally, Actinomycin D treatment showed that CASC19 stability was significantly enhanced in ALYREF-overexpressing RKO cells, whereas CASC19 stability was markedly reduced in sh-ALYREF cells (Fig. 5H).
To directly test whether ALYREF-mediated CASC19 stabilization depends on its m5C-recognition ability, we performed rescue experiments in sh-ALYREF RKO cells by reintroducing either wild-type ALYREF (ALYREF-wt) or an m5C-binding-domain mutant (ALYREF-mut). Western blot confirmed comparable expression levels of ALYREF-wt and ALYREF-mut (Fig. S1A–B). Functional assays demonstrated that ALYREF-mut cells exhibited significantly reduced proliferation compared with ALYREF-wt cells (CCK8, Fig. S1C). RNA stability assays showed that ALYREF-mut failed to restore CASC19 stability (Fig. S1D). Consistently, ALYREF-mut cells displayed impaired invasion and sphere-forming abilities relative to ALYREF-wt cells (Fig. S1E–F). These results provide direct evidence that ALYREF stabilizes CASC19 in an m5C-dependent manner, thereby promoting oncogenic phenotypes in CRC cells.
To further examine whether ALYREF promotes CASC19 stability through NSUN2-mediated m5C modification, we knocked down NSUN2 in ALYREF-overexpressing RKO cells and ALYREF-knockdown HCT116 cells. The results showed that CASC19 expression (Fig. 5I) and stability (Fig. 5J) were significantly altered. Further analysis revealed that CASC19 m5C modification levels were significantly increased in NSUN2-overexpressing cells (Fig. 5K), whereas these levels were significantly decreased upon NSUN2 knockdown (Fig. 5L). Moreover, NSUN2 overexpression enhanced CASC19 binding to ALYREF (Fig. 5M), while NSUN2 knockdown reduced their interaction (Fig. 5N). Collectively, these findings indicate that ALYREF directly binds CASC19 and promotes its m5C modification via NSUN2, which in turn stabilizes CASC19 expression. The ALYREF–NSUN2–CASC19 axis highlights a critical mechanism by which m5C modification regulates lncRNA stability and CRC progression.

CASC19 cooperates with ALYREF to promote CRC cell proliferation, survival, migration, invasion, and stemness
The findings above indicate that CASC19 is a downstream target of ALYREF, yet its role in tumor progression remains incompletely understood. To address this, we first silenced CASC19 in ALYREF-overexpressing RKO cells (Fig. 6A). This led to a significant decrease in cell viability (Fig. 6B) and a marked reduction in stemness markers, including CD133, CD44, SOX2, and ALDHA1 (Fig. 6C). Additionally, colony formation ability (Fig. 6D), migration, and invasion capacity (Fig. 6E) were all diminished, suggesting that CASC19 knockdown negatively regulates CRC cell aggressiveness. More importantly, CASC19 depletion resulted in increased apoptosis (Fig. 6F), cell cycle arrest (Fig. 6G), and reduced tumor sphere formation ability (Fig. 6H). These results demonstrate that CASC19 downregulation suppresses CRC cell proliferation and metastasis while promoting apoptosis.

Conversely, in ALYREF-knockdown HCT116 cells, we overexpressed CASC19 (Fig. 7A) and observed the opposite biological effects. Cell viability increased (Fig. 7B), along with upregulation of stemness-related markers CD133, CD44, SOX2, and ALDHA1 (Fig. 7C). Moreover, colony formation ability (Fig. 7D), migration, and invasion capacity (Fig. 7E) were significantly enhanced. Additionally, apoptosis was reduced (Fig. 7F), cell cycle progression was accelerated (Fig. 7G), and tumor sphere formation ability increased (Fig. 7H). These changes highlight CASC19’s role in promoting tumor cell proliferation and migration. In summary, our study demonstrates that CASC19 overexpression counteracts ALYREF knockdown effects in CRC cells, reinforcing the oncogenic role of ALYREF-CASC19 signaling in CRC progression.

CASC19 interacts with HNRNPC
CASC19 is a non-coding RNA, and its precise mechanism remains to be fully elucidated. By integrating multiple databases, including ENCORI, RNAInter, POSTAR3, TCGA, and GSE211831, we identified HNRNPC as a potential interacting protein of CASC19 (Fig. 8A). Further analysis showed that HNRNPC is highly expressed in TCGA-COAD and TCGA-READ datasets (Fig. 8B). Correlation analysis additionally demonstrated a significant positive association between HNRNPC and CASC19 expression levels in CRC samples (R = 0.26, Fig. S2A). Moreover, elevated HNRNPC expression was associated with poor prognosis in CRC patients (Fig. 8C), suggesting that HNRNPC, as an RNA-binding protein, may play an important role in CRC progression. To experimentally validate the interaction between CASC19 and HNRNPC, RNA pull-down and RIP assays were performed, which confirmed a direct binding between CASC19 and HNRNPC (Fig. 8D and E). To further map the interacting region, truncation experiments revealed that the F1 fragment of CASC19 is responsible for binding to HNRNPC (Fig. 8F). Consistently, RNA pull-down assays demonstrated that HNRNPC failed to bind to the CASC19-F1-mut construct, in which the HNRNPC-binding region was disrupted (Fig. S2B), further confirming the specificity of this interaction. Despite multiple validations of their interaction, overexpression or knockdown of CASC19 did not alter HNRNPC protein levels (Fig. 8G and H). This suggests that the interaction between CASC19 and HNRNPC may not regulate HNRNPC expression directly but could instead influence HNRNPC’s downstream molecular targets, thereby promoting CRC cell biological behaviors.

CASC19 recruits HNRNPC to regulate the mRNA stability of SCD in CRC
To further investigate the downstream molecular targets of the CASC19-HNRNPC axis, we conducted an intersection analysis of ENCORI, RNAInter, POSTAR3, TCGA (logFC > 2.9), and GSE211831 (logFC > 4.1) datasets, identifying SIM2, UBE2C, SCD, and VSNL1 as potential HNRNPC target genes (Fig. 9A). Subsequent bioinformatics analysis only indicated that high expression of SCD in CRC is associated with poor prognosis (Fig. 9B and C), suggesting a critical role of SCD in CRC progression, potentially under HNRNPC regulation. To explore the interaction between HNRNPC and SCD, we performed RNA pull-down and RIP assays, confirming their direct binding (Fig. 9D and E). Further experiments demonstrated that in RKO cells, HNRNPC overexpression increased SCD expression, while in HCT116 cells, HNRNPC knockdown significantly reduced SCD expression (Fig. 9F and H). These findings, combined with Fig. 8, suggest that the CASC19-HNRNPC axis may regulate CRC cell biological characteristics by modulating SCD mRNA levels.

To examine whether CASC19 affects SCD mRNA stability, we conducted stability assays. Overexpression of CASC19 in RKO cells significantly increased SCD mRNA stability, an effect that was reversed upon SCD knockdown using si-SCD (Fig. 9I). Conversely, CASC19 knockdown in HCT116 cells reduced SCD mRNA stability, but this reduction was partially restored upon SCD re-expression (Fig. 9I). To further determine whether the stabilizing effect of CASC19 on SCD mRNA depends on its HNRNPC-binding capacity, we employed a CASC19 F1 domain–deleted mutant (CASC19-F1-mut). When CASC19-F1-mut was co-transfected with si-SCD, SCD mRNA stability was significantly decreased compared with cells overexpressing wild-type CASC19 (CASC19-OE) (Fig. S2C–E). These findings indicate that the ability of CASC19 to stabilize SCD mRNA requires its intact F1 domain, which is responsible for HNRNPC binding. Collectively, these results provide strong evidence that CASC19 plays a critical role in maintaining SCD mRNA stability. Notably, despite the confirmed interaction between CASC19 and HNRNPC, no direct binding between CASC19 and SCD was detected (Fig. 9J). This observation suggests that CASC19 does not stabilize SCD mRNA through direct RNA–RNA interaction; rather, CASC19 likely functions as a molecular scaffold that recruits HNRNPC to SCD mRNA, thereby enhancing its stability and ultimately promoting colorectal cancer progression.

SCD overexpression reverses the suppressive effects of CASC19 knockdown on CRC cell proliferation, colony formation, cell cycle, apoptosis, migration, invasion, and stemness
SCD, a key enzyme in fatty acid metabolism, has gained increasing attention in CRC research. To investigate its role in CRC, we knocked down SCD and examined its impact on cellular phenotypes (Fig. 10A and B). The results showed that SCD knockdown significantly reduced cell viability (Fig. 10C, Fig. S2F) and downregulated key stemness markers including CD133, CD44, SOX2, and ALDHA1 (Fig. 10D). Moreover, colony formation ability was markedly reduced (Fig. 10E), along with significant suppression of cell migration and invasion (Fig. 10F, Fig. S2G). Additionally, apoptosis was increased (Fig. 10G), and cell cycle progression was inhibited (Fig. 10H), indicating that SCD knockdown suppresses CRC cell growth and expansion. Furthermore, tumorsphere formation ability was significantly decreased (Fig. 10I, Fig. S2H), highlighting the critical role of SCD in maintaining CRC cell stemness.

To further investigate the function of SCD in CRC, we overexpressed SCD in sh-CASC19 HCT116 cells (Fig. 11A and B). In contrast to the knockdown experiments, SCD overexpression significantly enhanced cell viability (Fig. 11C) and increased the expression of CD133, CD44, SOX2, and ALDHA1 (Fig. 11D). Furthermore, colony formation ability was restored (Fig. 11E), and cell migration and invasion were significantly promoted (Fig. 11F), suggesting that SCD overexpression enhances tumor metastasis potential. Additionally, apoptosis was reduced (Fig. 11G), and cell cycle progression was accelerated (Fig. 11H), indicating that SCD overexpression supports CRC cell survival and proliferation. Moreover, tumorsphere formation ability was significantly enhanced (Fig. 11I), confirming that SCD promotes CRC cell stemness. Collectively, these results suggest that SCD overexpression reverses the suppressive effects of CASC19 knockdown on CRC cell proliferation, colony formation, cell cycle progression, apoptosis, migration, invasion, and stemness. These findings underscore the crucial role of SCD in the CASC19-mediated regulation of CRC cell biological characteristics.

Discussion
This study systematically reveals the critical role of ALYREF in regulating RNA stability through m5C modification in CRC. As a key regulator of mRNA nuclear export and m5C modification [23], ALYREF overexpression is significantly associated with poor prognosis in CRC patients (Fig. 1N). This finding is consistent with previous reports [18], which also observed high ALYREF expression in CRC tissues. However, this study further elucidates its oncogenic mechanism. By integrating data from TCGA, GEO, and functional experiments, we discovered that ALYREF enhances the stability of CASC19 through NSUN2-mediated m5C modification (Fig. 5F-K). m5C modification has been shown to promote RNA nuclear–cytoplasmic transport and translation efficiency through recognition by reader proteins such as ALYREF [13, 24]. Notably, the synergistic association between ALYREF and NSUN2 (Fig. 1B) highlights a coordinated “writer–reader” regulatory mode, in which NSUN2 deposits m5C marks on target RNAs, while ALYREF selectively recognizes these modifications to promote RNA stabilization and sustained functional output. Such cooperation may enhance the specificity and efficiency of m5C-dependent gene regulation, ensuring persistent expression of oncogenic transcripts such as CASC19. From a translational perspective, disruption of this synergistic axis may represent a promising therapeutic strategy, as simultaneous interference with m5C deposition and recognition could more effectively attenuate RNA-driven malignant phenotypes in CRC. Together, these findings expand the functional scope of ALYREF within the epitranscriptome and provide a theoretical basis for targeting the m5C regulatory network in colorectal cancer.
For the first time, this study identifies the lncRNA CASC19 as a downstream effector molecule of the ALYREF/m5C axis and reveals its molecular pathway through the recruitment of HNRNPC to regulate SCD. While CASC19 overexpression is associated with poor prognosis in CRC [21], its regulatory mechanism has long been unknown. Our study demonstrates that CASC19 contains an NSUN2-dependent m5C modification (Fig. 5K, L), which significantly enhances its binding ability with ALYREF (Fig. 5M-N). This finding is consistent with recent studies showing that m5C modifications influence protein binding by regulating the secondary structure of lncRNAs [25]. Moreover, CASC19 specifically recruits HNRNPC through its F1 domain (Fig. 8F), contrasting with the mechanism by which HNRNPC binds m6A-modified lncRNAs to promote metastasis in cervical cancer [26]. This suggests that different RNA modifications may regulate RNA-protein interactions through spatial hindrance effects. These findings enrich the theoretical framework of lncRNA epitranscriptome regulation.
Additionally, this study establishes a functional link between the fatty acid metabolism enzyme SCD and the RNA-binding protein HNRNPC, unveiling the mechanism by which CASC19 drives CRC lipid metabolic reprogramming through stabilizing SCD mRNA. As the rate-limiting enzyme for monounsaturated fatty acid synthesis, SCD overexpression increases membrane fluidity and activates the PI3K/AKT pathway [27, 28]. This study further reveals that SCD expression is regulated by the HNRNPC-RNA complex (Fig. 9D–E). Notably, although HNRNPC does not directly alter SCD mRNA abundance (Fig. 9G, H), it enhances its stability (Fig. 9I), which is similar to the mechanism by which HNRNPC stabilizes IRAK1 mRNA in glioma [29]. Moreover, SCD overexpression significantly restores tumor spheroid formation capacity (Fig. 11I), suggesting that it maintains tumor stemness by regulating the lipid microenvironment. This is complementary to the reported activation of the Wnt/β-catenin pathway induced by SCD in liver cancer [30], collectively supporting the central role of lipid metabolic reprogramming in maintaining tumor stemness.
The ALYREF/m5C/CASC19/HNRNPC/SCD regulatory axis identified in this study provides multiple targets for precision therapy in CRC (Fig. S3). First, ALYREF inhibitors may exert therapeutic effects by disrupting m5C modification complexes. Second, CASC19-targeting ASO drugs or the CRISPR-dCas13b system could specifically block its interaction with HNRNPC. Furthermore, SCD inhibitors (e.g., MK-8245), which have shown anti-tumor activity in clinical trials, may be more effective when combined with the molecular subtyping outlined in this study. However, several questions remain to be addressed: (1) Does m5C modification cooperate with other reader proteins, such as YBX1, in regulating CASC19 function? (2) How do lipid metabolites (such as oleic acid) regulated by SCD specifically affect tumor stemness? (3) What is the role of the ALYREF-CASC19 axis in the tumor microenvironment (e.g., CAFs, TAMs)? (4) While our data indicate that ALYREF stabilizes NSUN2-mediated m5C-modified CASC19, we have not directly tested whether ALYREF and NSUN2 physically interact or influence each other’s recruitment to RNA. Future studies utilizing single-cell sequencing and spatial transcriptomics will help clarify the cellular heterogeneity of this pathway and drive the development of personalized CRC treatments.

Conclusion
This study systematically reveals the molecular mechanism by which ALYREF regulates the stability of lncRNA CASC19 through an m5C modification-dependent mechanism, and further drives the malignant progression of CRC through the CASC19-HNRNPC-SCD signaling axis. Specifically, (1) ALYREF is aberrantly overexpressed in CRC tissues and cell lines, and its expression level is significantly correlated with TNM staging and poor prognosis in patients, suggesting its potential as a diagnostic biomarker and prognostic indicator for CRC; (2) ALYREF recruits the m5C methyltransferase NSUN2 to mediate m5C modification in the 3’UTR region of CASC19, enhancing its binding to ALYREF and promoting mRNA stability, thus establishing the ALYREF/m5C/CASC19 regulatory axis; (3) CASC19 specifically recruits the RNA-binding protein HNRNPC through its F1 domain, forming a functional complex to enhance the stability of SCD mRNA, which in turn promotes CRC cell proliferation, metastasis, and stem cell characteristics through SCD-mediated lipid metabolic reprogramming; (4) In vitro and in vivo experiments confirm that targeted inhibition of ALYREF or CASC19 significantly suppresses tumor growth and lung metastasis, while restoring SCD expression reverses these phenotypes, validating the biological significance of this signaling pathway. These findings not only establish the first link between m5C modification and lncRNA-driven metabolic reprogramming but also highlight the core regulatory role of the ALYREF-CASC19-HNRNPC axis in CRC, providing new directions for developing RNA epigenetic regulation-based targeted therapeutic strategies.

Supplementary Information
Below is the link to the electronic supplementary material.