YTHDF1 enhances ACSL3 translation in an m6A-dependent manner to regulate lipid metabolism and colorectal cancer progression

Introduction

Rectal adenocarcinoma (READ) refers to a type of malignancy that develops within the gastrointestinal tract. Representing one of the predominant forms of colorectal cancer, it comprises about 30% of total cases (1). Rectal cancer possesses significant metastatic potential, which accounts for its high morbidity and mortality rates (2,3). Elderly populations are particularly vulnerable to this cancer (4), which ranks among the leading causes of cancer-associated death in this group (5). Despite considerable advances in treatment modalities—including laparoscopic surgery (6), minimally invasive therapy (7), adjuvant radio-chemotherapy, and immunotherapy—the outlook for READ patients remains unfavorable, characterized by diminished overall survival, especially among those presenting with advanced or metastatic conditions (8,9). Consequently, unraveling the molecular mechanisms driving READ and discovering innovative therapeutic targets represent an urgent research priority.

N⁶-methyladenosine (m⁶A) represents the most prevalent internal modification in eukaryotic messenger ribonucleic acid (mRNA), influencing nearly every stage of RNA metabolism, including splicing, degradation, export, and translation (10-14). A multiprotein writer complex, comprising Methyltransferase-like 3 (METTL3), Methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associating protein (WTAP), catalyzes the addition of m⁶A methylation, wherein METTL3 serves as the core catalytic component (15). On the other hand, the demethylases fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) act as erasers that eliminate m⁶A modifications from RNA transcripts. This reversible and dynamic attribute of m⁶A modification is governed by the balanced activities of writer and eraser enzymes (16,17). Additionally, several YTH domain-containing proteins—such as N⁶-methyladenosine RNA binding protein 1/2/3 (YTHDF1/2/3), YTH domain containing 1/2, and the insulin-like growth factor 2 mRNA-binding protein 1/2/3 (IGF2BP1/2/3) family—are identified as readers that selectively bind m⁶A sites and influence the fate of modified mRNAs (18,19). Growing evidence supports the involvement of m⁶A in a wide range of biological processes, including tissue morphogenesis, maintenance of embryonic stem cell pluripotency, and cell differentiation (20,21). Importantly, dysregulation of m⁶A modification has been strongly linked to the initiation and progression of numerous cancers, such as lung and liver cancers, as well as acute myeloid leukemia (22,23). Notably, the m⁶A regulatory network can play opposing roles in tumorigenesis, acting as either an oncogenic driver or a tumor suppressor in a context-dependent manner (24,25).

Lipid biosynthesis furnishes vital constituents for cellular membranes and supplies crucial metabolic intermediates required for cellular growth, division, and proliferation. Compared to normal counterparts, cancer cells display markedly enhanced proliferative potential and consequently demand accelerated lipid production to preserve membrane integrity and sustain rapid expansion, division, and multiplication (26-28). Acyl-CoA synthetase long chain family member 3 (ACSL3) contributes to the conversion of free long-chain fatty acids into fatty acyl-CoA esters, which is a process important for lipid synthesis and related metabolic activities (29).

In this study, analysis of The Cancer Genome Atlas (TCGA)-READ dataset revealed elevated YTHDF1 expression in READ specimens. This study investigated how the m⁶A reader protein YTHDF1 promotes READ progression by enhancing ACSL3 translation and influencing lipid metabolism. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0026/rc).

Methods

Cell culture

Human READ cell lines (SW837, SW1463, HR8348) and the normal fetal human colon epithelial cell line fetal human colon (FHC) were obtained from ATCC (City, USA). All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco, City, USA), under a humidified atmosphere of 5% carbon dioxide at 37 ℃.

Plasmids

To knock down YTHDF1 or ACSL3 expression, short hairpin RNA (shRNA) oligonucleotides were designed, annealed, and cloned into the pLKO.1 lentiviral vector (#10878, Addgene, City, USA). For ectopic expression, the full-length coding sequences of YTHDF1 or ACSL3 were inserted into the pCDH-CMV-MCS-EF1-copGFP lentiviral vector (#CD511B-1, System Biosciences, City, USA). Wild-type YTHDF1 and its mutant version (K395A, Y397A) were expressed from constructs based on the pCMV6 backbone (OriGene, City, USA). Additionally, HA-tagged ACSL3 and its mutant derivatives were generated by subcloning into the pcDNA3.1 expression vector (IDOBIO, City, China).

Cell transfection

The transient transfection of SW1463 cells was performed with plasmid vectors using the Lipofectamine 3000 reagent provided by Invitrogen (City, USA). To produce lentiviral particles, HEK293T cells (ATCC) were co-transfected with lentiviral transfer plasmids together with the packaging plasmids psPAX2 (#12260, Addgene) and pMD2.G (#12259, Addgene), employing Lipofectamine LTX (Invitrogen) as the transfection reagent. The viral supernatant was collected 48 hours after transfection, passed through 0.45 µm polyvinylidene fluoride (PVDF) filters, and used to transduce SW1463 cells.

Cell Counting Kit-8 (CCK-8) assay

Cell proliferation was assessed using the CCK-8 assay (Dojindo, City, Japan). SW1463 cells were seeded at 4,000 cells per well in 96-well plates and cultured for 24, 48, and 72 hours. At each time point, 10 µL of CCK-8 solution was added, followed by a 2-hour incubation at 37 ℃. Absorbance was then measured at 450 nm using a microplate reader (Thermo Fisher Scientific, City, USA).

Colony formation assay

SW1463 cells were plated at a density of 2,000 cells per well in 6-well plates and maintained in a medium supplemented with 10% FBS for 14 days. Following this incubation period, the cells were fixed using 4% paraformaldehyde and subsequently stained with 0.1% crystal violet. The colonies were then visualized and enumerated utilizing a Nikon light microscope (City, Japan).

Flow cytometry

Cells were harvested, fixed with 75% ethanol, stored at 4 ℃ overnight, and subsequently re-suspended in 500 µL of phosphate-buffered saline (PBS). Next, the cells were stained with propidium iodide (PI) and incubated in the dark for 30 minutes. The cell cycle analysis was performed using the BD Accuri C6 instrument (BD Biosciences, City, USA).

Cell apoptosis was detected with the annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Kit (Beyotime, City, China). Cells were collected and incubated with 5 µL of annexin V-FITC and 5 µL of PI in the dark for 15 minutes at 25 ℃ after resuspension. Next, the samples were analyzed by flow cytometry (BD Biosciences).

Wound healing assay

Cells were cultured in 6-well plates at 1×10⁵ cells per well until fully confluent. A wound was created with a sterile pipette tip, and the cells were washed with PBS to remove detached cell debris, floating dead cells and residual impurities, and to ensure a clear and regular scratch boundary for subsequent analysis. After washing, the cells were incubated for 24 hours. Images were captured at 0 and 24 hours to assess wound closure using ImageJ software (National Institutes of Health, City, USA).

Transwell assay

Cell invasive capacity was evaluated employing Transwell chambers pre-coated with Matrigel (BD Biosciences). A suspension of 1×10⁵ cells in 200 µL serum-free medium was added to the upper compartment. The lower chamber was filled with 600 µL of complete medium containing 10% FBS, which functioned as a chemoattractant. Following 24 hours of incubation, non-invading cells on the upper surface were gently removed. Cells that had invaded to the lower side were fixed in 4% paraformaldehyde, stained with 0.1% crystal violet, and quantified by microscopic examination.

Measurement of intracellular cholesterol (CH), triglyceride (TG), and adenosine triphosphate (ATP)

Intracellular levels of CH, TG, and ATP were measured to evaluate metabolic activity related to lipids and energy. For this purpose, 1×10⁶ cells were collected for analysis. Quantification of CH (MAK043, Sigma-Aldrich, City, USA) and TG (MAK266, Sigma-Aldrich) was performed with commercial detection kits in accordance with the provided protocols. ATP levels were assessed using a dedicated ATP Assay Kit (S0026, Beyotime). To enable consistent cross-sample comparisons, all obtained CH, TG, and ATP measurements were standardized against the corresponding sample’s total protein concentration.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted using TRIzol (Invitrogen) as per instructions. cDNA was synthesized with a Reverse Transcription Kit (TaKaRa, City, China). Gene expression was measured by RT-qPCR, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the control. Amplification occurred on an ABI 7500 (Applied Biosystems, City, USA) with these conditions: 94 ℃ for 30 s, followed by 40 cycles of 55 ℃ for 30 s and 72 ℃ for 90 s. Relative expression was calculated using the 2^−ΔΔCt method, with primer sequences in Table 1.

Western blot

Protein was extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) and quantified with a bicinchoninic acid assay kit (Pierce, City, USA). Equal protein amounts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were blocked for 1.5 hours at room temperature with 5% non-fat dry milk. Subsequently, the membranes were incubated overnight at 4 ℃ with specific primary antibodies against YTHDF1 (1:1,000; 43123, Cell Signaling Technology, City, USA), ACSL3 (1:1,000; 83319, Cell Signaling Technology), and GAPDH (1:3,000; 2118, Cell Signaling Technology). After thorough washing, the blots were incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5,000; 7074, Cell Signaling Technology). Immunoreactive bands were detected using an enhanced chemiluminescence system (Pierce), and GAPDH expression was used as an internal loading control.

RNA immunoprecipitation (RIP)

RIP assays were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, City, USA). Antibody coupling was achieved by incubating protein A/G magnetic beads with 10 µL of anti-YTHDF1 antibody or control IgG (Cell Signaling Technology) for 4 hours at 4 ℃. Cell lysates were then added to the antibody-bound beads and incubated overnight at 4 ℃. Immunoprecipitated RNA was isolated with TRIzol reagent (Invitrogen), and transcript enrichment levels were determined by RT-qPCR.

CrossLinking immunoprecipitation (CLIP)

CLIP was performed according to a previously described protocol (30). Briefly, 2×10⁷ cells were irradiated at 254 nm using a UV crosslinker (Thermo Fisher Scientific). Cell lysates were prepared using RIPA buffer and then incubated with protein A/G magnetic beads for 4 hours at 4 ℃. Immune complexes were eluted with 50 mM Tris-HCl (pH 7.8) at 60 ℃ for 20 minutes. RNA was extracted from the immunoprecipitates using chloroform and analyzed by RT-qPCR to evaluate target gene expression.

m⁶A immunoprecipitation (m⁶A-IP)

m⁶A-IP was conducted to isolate RNA fragments containing m⁶A modifications. Protein A/G magnetic beads were incubated overnight at 4 ℃ with 1.5 µg of anti-m⁶A antibody or control IgG. The antibody-bound beads were then incubated with 300 µg of fragmented RNA in RNase inhibitor-supplemented IP buffer overnight at 4 ℃. After immunoprecipitation, m⁶A-bound RNA was recovered using elution buffer and further purified by phenol/ethanol extraction. The purified RNA was then subjected to RT-qPCR for subsequent analysis.

Polysome profiling

Polysome profiling was performed in accordance with a previously established protocol (31). First, cells were treated with cycloheximide at a concentration of 0.1 mg/mL for 10 minutes, rinsed with ice-cold PBS, and then lysed using an extraction buffer. This buffer contained 10 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.3 M NaCl, 1% Triton X-100, RNase inhibitor, and 100 µg/mL cycloheximide. Afterward, a two-step centrifugation procedure was conducted: initial centrifugation at 2,000 rpm for 5 minutes, followed by centrifugation at 12,000 rpm for 15 minutes. Following this centrifugation process, RNA concentration was determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific). Next, 500 µg of RNA was loaded onto 10–50% sucrose density gradients and subjected to ultracentrifugation at 30,000 rpm for 4 hours at 4 ℃. Sequential fractions of 1 mL each were collected, and RNA was purified from every fraction using the RNeasy Mini Kit (QIAGEN, City, USA); the purified RNA was then used for subsequent RT-qPCR analysis.

Mouse xenograft experiments

Female BALB/c nude mice (5 weeks old) sourced from Guangdong Medical Laboratory Animal Center were subcutaneously inoculated with SW1463 cells that had been transduced with either sh-NC or sh-YTHDF1 lentiviral constructs. Tumor dimensions were measured every seven days using a caliper, and volumes were calculated as V = 0.5 × length × width². All animals were euthanized 28 days post-inoculation. Experiments were performed under a project license (No. the license number) granted by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University (Xiamen Branch), in compliance with national guidelines for the care and use of animals.

TCGA data analysis

YTHDF1 expression profiles were acquired from the TCGA repository. Differential expression of YTHDF1 and ACSL3 between normal tissues and READ samples was assessed via Student’s t-test. The association between YTHDF1 and ACSL3 expression levels within READ tissues was examined using Spearman’s rank correlation analysis.

Statistical analysis

Statistical analyses were conducted with SPSS Statistics 22.0 (IBM Corp., City, USA) and GraphPad Prism 10.0 (GraphPad Software Inc., City, USA). Data are presented as mean ± standard deviation. The differences between the two groups were evaluated utilizing the Student’s t-test. For analyses involving three or more groups, a one-way analysis of variance was conducted, followed by Tukey’s post hoc test for multiple comparisons. A P value of less than 0.05 was regarded as indicative of statistical significance.

Results

YTHDF1 is highly expressed in READ

Initial screening of m⁶A regulatory factors in the TCGA-READ dataset aimed to identify proteins potentially involved in READ. The analysis revealed a statistically significant elevation in YTHDF1 expression levels in READ tissues compared to normal tissues (Figure 1A). Subsequent evaluation of YTHDF1 expression in READ cell lines (SW837, SW1463, HR8348) demonstrated consistent upregulation at both mRNA and protein levels, as determined by RT-qPCR and Western blot analyses. The most pronounced upregulation was observed in SW1463 cells (Figure 1B,1C), leading to the selection of this cell line for subsequent experimental investigations.

Figure 1 YTHDF1 is highly expressed in READ. (A) YTHDF1 expression in the TCGA-READ dataset. (B,C) YTHDF1 expression in READ cell lines detected by RT-qPCR and Western blot. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. FHC, fetal human colon; N, READ, rectal adenocarcinoma; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation; T; TCGA, The Cancer Genome Atlas; TPM.

Downregulation of YTHDF1 suppresses proliferation, migration, invasion, and lipid synthesis in READ cells

To investigate the biological role of YTHDF1 in READ, SW1463 cells were transfected with knockdown or overexpression constructs targeting YTHDF1. Verification of the transfection’s effectiveness was achieved through RT-qPCR and Western blot analyses (Figure 2A,2B). According to CCK-8 assay results, suppression of YTHDF1 expression markedly reduced cellular viability (Figure 2C), and colony formation capacity was also impaired after YTHDF1 knockdown (Figure 2D). Analysis of cell cycle distribution revealed more cells were halted in the Gap 0 (G0)/Gap 1 (G1) phase and apoptosis increased after YTHDF1 was silenced (Figure 2E,2F). Wound healing was delayed in cells with reduced YTHDF1 expression (Figure 3A), and Transwell invasion assays demonstrated a decline in invasive ability (Figure 3B). Additionally, depletion of YTHDF1 resulted in lowered intracellular concentrations of CH, TG, and ATP (Figure 3C-3E). Conversely, overexpression of YTHDF1 enhanced proliferative, migratory, invasive, and lipid metabolic activities. Collectively, these findings demonstrate that downregulation of YTHDF1 restrains malignant behaviors and lipid biosynthesis in READ cells.

Figure 2 Downregulation of YTHDF1 suppresses proliferation in READ cells. (A,B) Validation of YTHDF1 knockdown and overexpression by RT-qPCR and Western blot. (C) Cell viability measured by CCK-8 assay. (D) Cell proliferation assessed by colony formation assay. (E,F) Cell cycle and apoptosis analyzed by flow cytometry. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; NC; OD; oe, PI, propidium iodide; READ, rectal adenocarcinoma; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation; sh.

Figure 3 Downregulation of YTHDF1 suppresses migration, invasion, and lipid synthesis in READ cells. (A) Cell migration evaluated by wound healing assay. (B) Cell invasion detected by Transwell assay. (C-E) Intracellular levels of CH, TG, and ATP. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ATP, adenosine triphosphate; CH, cholesterol; NC, oe; READ, rectal adenocarcinoma; SD, standard deviation; sh, TG, triglyceride.

Downregulation of YTHDF1 suppresses tumor growth in vivo

To evaluate the impact of YTHDF1 on READ tumor growth in vivo, SW1463 cells expressing sh-NC or sh-YTHDF1 were subcutaneously injected into BALB/c nude mice. Tumor progression was monitored over time, and results indicated that tumors derived from sh-YTHDF1-transduced cells showed markedly reduced volume and weight relative to those in the sh-NC group (Figure 4A-4C).

Figure 4 Downregulation of YTHDF1 suppresses tumor growth in vivo. (A) Representative images of xenograft tumors. (B) Tumor volume. (C) Tumor weight. Data are presented as mean ± SD. *, P<0.05; **, P<0.01. NC, SD, standard deviation; sh.

ACSL3 is an m⁶A-modified target gene of YTHDF1

Consistent with prior reports indicating that YTHDF1 modulates the translation of m⁶A-modified transcripts (32), analysis of the TCGA-READ dataset demonstrated significantly upregulated ACSL3 expression in READ tissues compared to normal tissues (Figure 5A). Moreover, ACSL3 expression levels showed a positive correlation with YTHDF1 expression in READ samples (Figure 5B). Bioinformatic prediction using RMBase v3.0 and ENCORI databases identified potential m⁶A modification sites and YTHDF1 binding motifs within ACSL3 mRNA (Figure 5C,5D). Experimental validation through RIP, CLIP, and m⁶A-IP assays confirmed the physical association between ACSL3 and YTHDF1, as well as the presence of m⁶A modifications on ACSL3 transcripts (Figure 5E-5G).

Figure 5 ACSL3 is an m⁶A-modified target gene of YTHDF1. (A) ACSL3 expression in the TCGA-READ dataset. (B) Correlation between YTHDF1 and ACSL3 expression in the TCGA-READ dataset. (C) m⁶A modification sites on ACSL3 mRNA predicted by RMBase v3.0. (D) YTHDF1 binding sites on ACSL3 mRNA predicted by ENCORI. (E) RIP assay. (F) CLIP assay. (G) m⁶A-IP assay. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. CLIP, CrossLinking immunoprecipitation; ENCORI, IgG, N, READ, rectal adenocarcinoma; RIP, RNA immunoprecipitation; SD, standard deviation; T, TCGA, The Cancer Genome Atlas; TPM.

YTHDF1 promotes ACSL3 translation in an m⁶A-dependent manner

To determine the regulatory role of YTHDF1 in ACSL3 expression at the mechanistic level, lentiviral-mediated knockdown (sh-YTHDF1) or overexpression (oe-YTHDF1) was performed in SW1463 cells, followed by RT-qPCR and Western blot analysis. No notable changes were observed in ACSL3 mRNA across groups; however, ACSL3 protein levels were markedly reduced under YTHDF1 depletion and elevated upon its overexpression (Figure 6A,6B). Polysome profiling indicated that suppression of YTHDF1 impaired, whereas its enhancement augmented, the translational efficiency of ACSL3 (Figure 6C). Moreover, transfection with wild-type YTHDF1 (YTHDF1-WT) elevated ACSL3 translation efficiency, an effect not observed with the mutant form (YTHDF1-MUT) (Figure 6D). These findings suggest that YTHDF1 facilitates ACSL3 translation in READ cells.

Figure 6 YTHDF1 promotes ACSL3 translation in an m⁶A-dependent manner. (A,B) ACSL3 expression detected by RT-qPCR and Western blot. (C,D) Polysome profiling analysis. (E) RIP assay. (F,G) ACSL3 expression detected by Western blot. Data are presented as mean ± SD. ns; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. IgG, MUT; NC, oe, RIP, RNA immunoprecipitation; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation; sh; WT.

To assess m⁶A dependency in this regulatory process, this study leveraged prior evidence that YTHDF1 recognizes m⁶A motifs via its YTH domain, with point mutations at K395 and Y397 abrogating RNA binding (33). SW1463 cells were transfected with FLAG-tagged YTHDF1-WT or a double mutant YTHDF1-MUT (K395A/Y397A). RNA immunoprecipitation confirmed strong enrichment of ACSL3 mRNA by YTHDF1-WT, which was substantially diminished with YTHDF1-MUT (Figure 6E). Concordantly, YTHDF1-WT—but not YTHDF1-MUT—upregulated ACSL3 protein expression (Figure 6F). Further experiments using HA-tagged ACSL3, either wild-type (ACSL3-WT) or bearing m⁶A site mutations (ACSL3-MUT), revealed that significant elevation of ACSL3 protein occurred exclusively in cells co-transfected with YTHDF1-WT and ACSL3-WT (Figure 6G). Collectively, these data establish that YTHDF1 enhances ACSL3 translation in an m⁶A-dependent fashion.

Downregulation of ACSL3 suppresses proliferation, migration, invasion, and lipid synthesis in READ cells

The functional impact of ACSL3 in READ cells was further examined. The transduction efficiency of ACSL3 knockdown or overexpression in SW1463 cells was validated by RT-qPCR and Western blot (Figure 7A,7B). Evaluation of cellular metabolic activity using CCK-8 assays indicated that suppression of ACSL3 led to a decline in cell viability (Figure 7C). Similarly, a reduction in proliferative capacity was observed in colony formation experiments following ACSL3 depletion (Figure 7D). Flow cytometry analysis revealed that knocking down ACSL3 caused cells to halt in the G0/G1 phase and increased apoptosis rates (Figure 7E,7F). Migration and invasion capabilities were also compromised under ACSL3 deficiency, as evidenced by impaired wound closure in healing assays and decreased penetration in Transwell experiments (Figure 8A,8B). Metabolic profiling revealed that ACSL3 knockdown significantly reduced intracellular concentrations of CH, TG, and ATP (Figure 8C-8E). Conversely, ACSL3 overexpression produced oncogenic effects, enhancing the aforementioned malignant phenotypes (Figures 7C-7F,8A-8E). These collective findings demonstrate that downregulation of ACSL3 inhibits proliferation, migration, invasion, and lipid synthesis in READ cells.

Figure 7 Downregulation of ACSL3 suppresses proliferation in READ cells. (A,B) Validation of ACSL3 knockdown and overexpression by RT-qPCR and Western blot. (C) Cell viability measured by CCK-8 assay. (D) Cell proliferation assessed by colony formation assay. (E,F) Cell cycle and apoptosis analyzed by flow cytometry. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; NC; OD, oe; PI, propidium iodide; READ, rectal adenocarcinoma; RT-qPCR, real-time quantitative polymerase chain reaction; SD, standard deviation; sh.

Figure 8 Downregulation of ACSL3 suppresses migration, invasion, and lipid synthesis in READ cells. (A) Cell migration evaluated by wound healing assay. (B) Cell invasion detected by Transwell assay. (C-E) Intracellular levels of CH, TG, and ATP. Data are presented as mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. ATP, adenosine triphosphate; CH, cholesterol; NC; oe; READ, rectal adenocarcinoma; SD, standard deviation; sh; TG, triglyceride.

Upregulation of ACSL3 attenuates the inhibitory effects of YTHDF1 downregulation on proliferation, migration, invasion, and lipid synthesis in READ cells

To analyze the interaction between YTHDF1 and ACSL3 in READ, ACSL3 was overexpressed in SW1463 cells with reduced YTHDF1 expression. The successful upregulation of ACSL3 protein was verified by Western blot analysis (Figure 9A). Phenotypic and metabolic evaluations were performed across three groups: sh-NC, sh-YTHDF1, and sh-YTHDF1 + oe-ACSL3, using CCK-8, colony formation, flow cytometry, wound healing, Transwell assays, and lipid metabolite quantification. The data demonstrated that ectopic expression of ACSL3 markedly reversed the inhibitory effects induced by YTHDF1 depletion on cell proliferation, migration, invasion, and lipid synthesis (Figures 9C-9E,10A-10E).

Figure 9 Upregulation of ACSL3 attenuates the inhibitory effects of YTHDF1 downregulation on proliferation in READ cells. (A) ACSL3 expression detected by Western blot. (B) Cell viability measured by CCK-8 assay. (C) Cell proliferation assessed by colony formation assay. (D,E) Cell cycle and apoptosis analyzed by flow cytometry. Data are presented as mean ± SD. *, P<0.05; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; FITC, fluorescein isothiocyanate; NC, OD; oe; PI, propidium iodide; READ, rectal adenocarcinoma; SD, standard deviation; sh.

Figure 10 Upregulation of ACSL3 attenuates the inhibitory effects of YTHDF1 downregulation on migration, invasion, and lipid synthesis in READ cells. (A) Cell migration evaluated by wound healing assay. (B) Cell invasion detected by Transwell assay. (C-E) Intracellular levels of CH, TG, and ATP. Data are presented as mean ± SD. **, P<0.01; ***, P<0.001. ATP, adenosine triphosphate; CH, cholesterol; NC, oe, READ, rectal adenocarcinoma; SD, standard deviation; sh, TG, triglyceride.

Discussion

READ is among the most prevalent malignant tumors affecting humans. Due to the more complex anatomical location and histological structure compared to colon cancer, READ poses significant challenges for clinical management (33,34). Currently, the primary treatment strategies for READ still heavily rely on conventional chemotherapy and radiotherapy, while targeted therapies widely used in other malignancies remain limited in this field. Therefore, identifying novel molecular targets and developing corresponding therapeutic approaches are crucial for the treatment of this disease.

Growing evidence indicates that m⁶A modification plays a key role in every hallmark of cancer biology (35). In this study, analysis of the TCGA-READ dataset revealed YTHDF1 as the most significantly upregulated m⁶A regulator in READ. Previous studies have demonstrated various functions of YTHDF1 in tumorigenesis, including promoting cancer cell proliferation (36,37), facilitating epithelial-mesenchymal transition (38,39), enhancing tumor immune escape (40,41), increasing chemotherapy resistance, and driving cell cycle progression (42). Moreover, the present study demonstrated that the downregulation of YTHDF1 inhibited the proliferation, migration, and invasion of READ cells, induced cell cycle arrest, and facilitated apoptosis. In addition, downregulation of YTHDF1 was shown to reduce tumor growth in vivo.

Lipid metabolism encompasses a series of intricate biochemical reactions, including the breakdown, uptake, biosynthesis, and catabolism of lipids mediated by diverse enzymes, all crucial for preserving cellular homeostasis. To support their heightened nutritional requirements, cancer cells frequently modulate and hijack lipid metabolic pathways to facilitate proliferation, survival, invasive capacity, and metastatic dissemination (43). Previous studies have shown that lipid metabolic reprogramming occurs in multiple tumor types (44,45). Furthermore, dysregulation of m⁶A modification has been confirmed to mediate lipid metabolic reprogramming in various cancers (46,47). In this study, knockdown of YTHDF1 reduced intracellular levels of CH, TG, and ATP, indicating that YTHDF1 downregulation suppresses lipid synthesis in READ cells.

ACSL3, as a key player in intracellular lipid metabolism, influences fatty acid activation and related metabolic processes (48). Previous research has indicated that ACSL3 is involved in the development of various cancer types (49-51). The present research discovered that ACSL3 has m⁶A binding sites, and further validation showed that YTHDF1 enhances ACSL3 translation in a manner dependent on m⁶A. Functional analyses demonstrated that downregulation of ACSL3 inhibits proliferation, migration, invasion, and lipid synthesis in READ cells. Moreover, upregulation of ACSL3 was shown to attenuate the inhibitory effects of YTHDF1 knockdown on these malignant phenotypes.

However, the limitations of this study should be acknowledged in subsequent research. A primary constraint is the absence of validation using clinical READ tissue specimens. Consequently, the expression pattern of YTHDF1 in patient-derived samples remains uncharacterized, and its potential associations with critical clinicopathological parameters—including patient age, tumor staging, and lymph node metastasis—were not explored. These omissions somewhat diminish the clinical translatability of the results and complicate the assessment of YTHDF1’s utility as a biomarker or therapeutic target in READ management.

Conclusions

In summary, this study provides novel evidence that YTHDF1 facilitates the progression of READ by promoting the translation of ACSL3 in a manner dependent on m⁶A modification, thereby enhancing lipid metabolism. These results enhance the understanding of m⁶A modification’s role in gastrointestinal cancers and offer a new theoretical and experimental basis for creating diagnostic biomarkers and targeted treatments for READ.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0026/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0026/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0026/prf

Funding: This work was supported by Guiding Medical and Health Project of Xiamen City, 2024 (grant No. 3502Z20244ZD1102), General Program of Fujian Provincial Natural Science Foundation (grant No. 2023J011688), Medical Innovation Project of Fujian Provincial Health Commission (grant No. 2021CXB024), and Foundation for Cultivated Young Talents of Fujian Province, China (No. 2025350998).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0026/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. the license number) granted by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University (Xiamen Branch), in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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