In total, three CRC cell lines (LS174T, HCT116 and SW480) and two normal human colon mucosal epithelial cell lines (NCM-460 and HIEC-6) were utilized in the present study, all acquired from The Cell Bank of Type Culture Collection of The Chinese Scientific Academy. The HCT116, SW480, NCM-460 and HIEC-6 cell lines were regularly maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator set at 37°C and 5% CO₂. Conversely, the LS174T cells were cultured using Dulbecco's Modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin.

To perform RNA interference, HCT116 and SW480 cells were seeded in 60 mm tissue culture plates for 24 h prior to transfection. METTL3 short hairpin (sh)RNA cloned into the pLKO.1 lentiviral vector (Addgene, Inc.) was obtained. Subsequently, the vector (pLKO.1-METTL3) was co-transfected into CRC cells with the psPAX2 and pmD2G (Addgene, Inc.) packing plasmids at a 4:3:1 ratio, using the PolyJet reagent (SignaGen Laboratories) in serum-free media. After 48 h transfection at 37°C, the cell culture supernatant was harvested and filtered through a 0.22 µm membrane. Following concentration using a 100 kD Millipore filter, the viral solution was added to the cancer cell culture medium at a multiplicity of infection of 3, allowing for a 24-h infection period. Successfully transfected cells expressing shRNAs were selected using 5 µg/ml puromycin (Sigma-Aldrich; Merck KGaA) and maintained in RPMI-1640/10% FBS supplemented with 1 µg/ml puromycin at 37°C and 5% CO₂. The sequences of the shRNAs were as follows: sh-METTL3, 5′-GCACTTGGATCTACGGAATCC-3′; and sh negative control (NC), 5′-GTCCATCGAACTCAGTAGCT-3′.

To achieve METTL3 overexpression, HCT116 cells were stably transfected with the pcDNA3/Flag-METTL3 plasmid (cat. no. 53739; Addgene, Inc.). HCT116 and SW480 cells were stably transfected with the pcDNA3-MMP9 plasmid (Suzhou Hongxun Biotechnologies Co., Ltd.). The transfection procedure was conducted utilizing Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Briefly, the cells were grown in 6-well plates until they reached 90% confluency. Then, the cells were transfected with plasmid DNA (5.0 mg per well), utilizing Lipofectamine 3000 (10 µl) to facilitate transfection at 37°C. After 48 h, the cells were subjected to trypsinization and subsequently transferred to 10-cm culture dishes in medium supplemented with 300 mg/ml G418 (Gibco; Thermo Fisher Scientific, Inc.) and maintained with 50 mg/ml G418. Clonal expansion was achieved through the isolation of single-cell clones.

Total RNA was isolated using the TRIzol reagent (Ambion; Thermo Fisher Scientific, Inc.). Subsequently, the polyadenylated RNA was extracted from the total RNA using the Dynabeads™ mRNA Purification Kit (Invitrogen; Thermo Fisher Scientific, Inc.), with any contaminating rRNA removed by the RiboMinus transcriptome isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.). The relative levels of m⁶A in the mRNA were assessed utilizing an m⁶A RNA Methylation Quantification Kit (Colorimetric; cat. no. P-9005; EpigenTek Group, Inc.), according to the manufacturer's instructions. Briefly, capture and detection antibodies were employed to measure the m⁶A levels, which were determined at measuring the absorbance at 450 nm. Each reaction was replicated three times to quantify the relative absorbance.

Total cellular RNA was isolated using the RNA isolation kit (Shanghai Yeasen Biotechnology Co., Ltd.) and subsequently reverse transcribed into cDNA with the PrimeScript™ RT Reagent Kit, which included a gDNA Eraser (Takara Bio, Inc.). qPCR was conducted using the TB Green^® Premix Ex Taq™ (Takara Bio, Inc.). The qPCR thermocycling conditions were as follows: 40 cycles at 37°C for 15 min, 60°C for 5 sec and 72°C for 30 sec. The relative mRNA expression levels were calculated using the 2^−∆∆Cq method (28). All procedures followed the manufacturer's instructions and were repeated three times. The primer (Sangon Biotech Co., Ltd.) sequences were as follows: METTL3 forward, 5′-CTATCTGGCACTCGCAAGA-3′ and reverse, 5′-GCTTGAACCGTGCAACCACATC-3′; MMP9 forward, 5′-CCAATCACCACCATCCGTTG-3′ and reverse, 5′-CCTCGGGCAAATGTCTTACC-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCAA-3′; and 18S forward, 5′-GGAGTATGGTTGCAAAGCTGA-3′ and reverse, 5′-ATCTGTCAATCCTGTCCGTGT-3′.

Stable knockdown METTL3 cell RNA was extracted, and polyadenylated RNA was enriched using an mRNA purification kit (Thermo Fisher Scientific, Inc.). This enriched RNA was then treated with DNase I (Thermo Fisher Scientific, Inc.). Following this, 100 µg global RNA was incubated with m⁶A or IgG antibodies for immunoprecipitation using the Magna methylated RNA immunoprecipitation (MeRIP) m⁶A kit (cat. no. 17-10499; MilliporeSigma) according to the manufacturer's instructions. For m6A RIP qPCR, the total RNAs were fragmented into 300-nucleotide fragments after incubation in fragmentation buffer at 94°C for 30 sec and immunoprecipitated using anti-m6A antibody according to the manufacturer's instructions. In total, one-tenth of the fragmented RNAs were saved as input control, and the enrichment of m6A was quantified using RT-qPCR.

To assess the degradation rate of MMP9 mRNA, cells were treated with Actinomycin-D (Act-D; Sigma-Aldrich; Merck KGaA) at a final concentration of 3 µg/ml at 37°C to inhibit new RNA synthesis. At specified intervals, cell samples were collected and analyzed using qPCR (as aforementioned). The MMP9 expression levels were standardized against 18S RNA.

To measure protein stability, shNC and shMETTL3 transfected cells were seeded into 6-well plates and treated with cycloheximide (Cayman Chemical Company; to inhibit RNA translation) at 37°C at final concentration of 20 µg/ml for the indicated times. Cells were collected and lysed in RIPA lysis buffer (Cell Signaling Technology, Inc.). The expression of proteins was measured through western blot analysis.

In total, 100 µl of culture medium containing 3×10⁴ cells were seeded into 96-well plates and cultured for 24 h. Subsequently, 10 µl Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories, Inc.) was added to each well. Following an additional 2-h incubation in a humidified environment at 37°C with 5% CO₂, the absorbance of the cells at 450 nm was measured using a spectrophotometer. Each assay was performed in triplicate.

To assess the migration capability, 1×10⁶ cells per well were cultured in 6-well plates until a 80–90% confluency was reached. A sterile 200 µl pipette tip was employed to create straight uniform scratches. Cells were serum-starved prior to and during the assay. Images were obtained by light microscopy (Nikon C1 Eclipse; Nikon Corporation) and captured at both 0 and 48 h, with each experimental condition tested in triplicate. Image analysis was performed using ImageJ (version1.42q; National Institutes of Health). The relative scratch width was expressed as: (original scratch width-new scratch width)/original scratch width ×100%.

For evaluating the invasion capability, Transwell chambers pre-coated with Matrigel (BD Biosciences) at 37°C for 4 h were utilized. The upper chamber contained the specific cells (1×10⁴ cells) in serum-free medium, while the lower chamber contained complete medium supplemented with 10% FBS. Cells that invaded the surface of the lower chamber after 24 h were then fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet (Beijing Solarbio Science & Technology Co., Ltd.) for 3 min at room temperature. After air drying, the invaded cells were imaged using a light microscope (Nikon C1 Eclipse; Nikon Corporation) and quantified by counting cells in five randomly selected fields. Image analysis was performed using ImageJ (version1.42q; National Institutes of Health).

To evaluate apoptosis, flow cytometry was employed utilizing a FITC-Annexin V/PI detection kit (Wanleibio, Co., Ltd.). Each well was seeded with 1×10⁶ cells in 6-well plates, followed by harvesting after 48 h of incubation at 37°C. The cells were then stained with FITC-Annexin V and PI for 15 min at room temperature, in the dark. Apoptotic cell percentages were subsequently determined through FACS flow cytometry (FACS Canton II; BD Biosciences) and the data were analyzed using FlowJo 8.6.3 (Tree Star, Inc.).

Cellular proteins were extracted utilizing RIPA buffer (cat. No. 9806; Cell Signaling Technology, Inc.) supplemented with a protease and phosphatase inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Protein concentrations were quantified using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). The proteins (20 µg per lane) were resolved on 10% SDS-PAGE gels and subsequently transferred PVDF membranes. To block non-specific binding, the membranes were incubated with 5% non-fat milk in TBST (0.1% Tween-20) for 2 h at room temperature. Following this, the membranes were incubated overnight at 4°C with primary antibodies against METTL3 (1:2,000; cat. no. 86132S; Cell Signaling Technology, Inc.), MMP9 (1:1,000; cat. no. ab76003; Abcam) and GAPDH (1:5,000; cat. no. 5174S; Cell Signaling Technology, Inc.). After three washes with PBST (0.1% Tween 20), the membranes were incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000; cat. no. BA1055; Boster Biological Technology). The signal was detected using ECL western blotting detection reagents (Tanon Science and Technology Co., Ltd.). Quantification of protein expression levels was performed using ImageJ software (version 1.50; National Institutes of Health).

HCT116, METTL3 knockdown HCT116 and METTL3 overexpressing HCT116 cells (5×10⁶ cells; n=5 mice per group) were suspended in 100 µl PBS and Matrigel (Shanghai Yeasen Biotechnology Co., Ltd.) at a 1:1 ratio. This cell mixture was then subcutaneously injected into the flanks of 4-week-old nude mice (weighing 14–16 g). All 15 mice were placed in SPF housing conditions with a 12:12-h light/dark cycle and at a constant temperature (22±2°C). Furthermore, the mice were given unrestricted access to standard chow and water. Throughout the experimental period, mice were monitored three times a week for tumor development. The tumors were measured using calipers, with a humane endpoint set at a tumor diameter of >2,000 mm. The tumor volume was calculated using the following formula: Volume=W² × L/2, where W represents the short diameter and L represents the long diameter. The weights of the mice were rigorously monitored to ensure that any decrease did not exceed 20%, thereby mitigating potential suffering. No animals died before meeting the criteria for humane endpoint euthanasia. At the end of the experiment (day 24), the mice were anesthetized through intraperitoneal administration of sodium pentobarbital (50 mg/kg) and subsequently sacrificed by cervical dislocation. Following euthanasia, the xenograft tumors were carefully excised from the sacrificed mice and weighed immediately. The experiments adhered to institutional guidelines and ethical standards regarding euthanasia and death verification. Some tumor specimens were used for RNA extraction with TRIzol, followed by qPCR to assess the METTL3 and MMP9 mRNA expression levels (as aforementioned). The remaining samples were fixed with 10% (v/v) neutral-buffered formalin for 24 h at room temperature, then transferred to 70% ethanol until they were embedded in paraffin and sectioned at a 3 µm thickness. For hematoxylin-eosin staining, the sections were treated with hematoxylin for 2 min and eosin for 1 min.

Antigen retrieval was performed using boiling sodium citrate buffer (0.1 M, pH 4). After deparaffinization and hydration of the paraffin-embedded tissue sections, endogenous peroxidase activity was blocked with Peroxidase 1 blocking reagent (Biocare Medical, LLC) for 10 min followed by blocking with Background Sniper serum-free blocking reagent (Biocare Medical, LCC) for 15 min at room temperature. Tissue sections were incubated overnight at 4°C with primary antibodies targeting METTL3 (1:50; cat. no. 86132S; Cell Signaling Technology, Inc.), MMP9 (1:1,000; cat. no. ab76003; Abcam) and Ki67 (1:100; cat. no. ab16667; Abcam). Post incubation, sections were washed with PBST (0.05% Tween-20) and then treated with a HRP-conjugated rabbit secondary antibody (1:5,000; cat. no. BM3894; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. The sections were subsequently developed using 0.05% 3-diaminobidine tetrahydrochloride for 10 sec at room temperature, followed counterstaining with 10% Mayer's hemoxylin for 4 min at room temperature. The IHC results were analyzed by two experienced pathologists. For imaging, five random fields at ×200 magnification under a light microscope (Leica DMI4000B; Leica Microbiosystems GmbH) were selected.

The Gene Expression Profiling Interactive Analysis database (gepia.cancer-pku.cn/) served as the tool to assess the mRNA expression levels of METTL3, METTL14, WTAP, ALKBH5 and FTO in both CRC and normal tissues. For Kaplan-Meier analysis, the log-rank test was employed. To determine the overall survival of patients with CRC and varying levels of METTL3 expression, Kaplan-Meier survival analyses were performed using the Kaplan Meier plotter online tool (http://kmplot.com/analysis/) with default parameters. The median value was set as the cut-off.

Each experiment was conducted a minimum of three times, with representative outcomes illustrated. Data are presented as the mean ± SD. One-way analysis of variance with Tukey's multiple comparisons was used to identify significant differences among three or more groups, while two groups were compared using the unpaired Student's t-test. Data analysis was performed using GraphPad Prism 8 software (Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of m⁶A modification in CRC in vitro, the m⁶A levels in three CRC cell lines (LS174T, HCT116 and SW480) and two normal human colon mucosal epithelial cell lines (HIEC-6 and NCM-460) were quantified using the colorimetric m⁶A quantification assay. The results demonstrated that the global mRNA m⁶A levels were elevated in the CRC cell lines compared with the normal cell lines (Fig. 1A). To assess the expression profiles of m⁶A writers and erasers in CRC, The Cancer Genome Atlas database was analyzed, and it was found that METTL3 was upregulated in CRC clinical tissues (Fig. 1B). The RT-qPCR results further indicated a significant increase in METTL3 expression in CRC cell lines, particularly in HCT116 and SW480 cells, compared with the normal epithelial cell lines (Fig. 1C). These findings suggest that METTL3, an RNA methyltransferase, may play a crucial role in CRC progression. Moreover, METTL3 expression was higher in advanced clinical stages than in the early stages, showing a significant positive association (Fig. 1D). Kaplan-Meier survival curves indicated that higher METTL3 expression was significantly associated with a lower survival rate among patients with CRC (Fig. 1E). Thus, these results indicate that METTL3 is highly expressed in CRC and is associated with poor prognosis.

To explore the role of METTL3 in the progression of CRC as suggested by the clinical data, the proliferation and invasion capabilities of CRC cells were examined. For this purpose, two stable CRC cell lines with METTL3 knockdown were established using different shRNAs delivered via lentivirus. Validation of METTL3 knockdown at the mRNA level showed a reduction of 25% in HCT116 cells and 45% in SW480 cells (Fig. 2A). Correspondingly, the METTL3 protein levels were significantly lower in the knockdown cells compared with the controls. Densitometric analysis confirmed a 20% decrease in METTL3 protein in HCT116 cells and a 43% decrease in SW480 cells (Fig. 2B). RT-qPCR and western blotting therefore demonstrated the notable METTL3 knockdown efficiency of the lentivirus used. The stable with/without METTL3 knockdown (shMETTL3 and shNC) HCT116 and SW480 cells were employed to investigate the influence of METTL3 on the proliferation and migration capacity. The CCK-8 assay results demonstrated a significant downregulation of the viability of the shMETTL3 HCT116 and SW480 cells (Fig. 2C). Additionally, the Transwell and scratch assays demonstrated that shMETTL3 cells exhibited reduced invasion (Fig. 2D) and migration (Fig. 2E) capacities compared with shNC cells. Flow cytometry further showed that METTL3 knockdown increased apoptosis in CRC cell lines (Fig. 2F). Overall, these results suggest that knocking down METTL3 decreases cell viability and migration while promoting apoptosis in CRC cell lines.

MMP9 plays a promotive role in various cancer types; DNA methylation of the MMP9 gene has been shown to result in the upregulation of MMP9 protein expression, thereby facilitating cancer progression and metastasis (29). We hypothesized that METTL3 may regulate the expression of MMP9 in an m⁶A-dependent manner, further participating in CRC tumorigenesis via post-transcriptional regulation. The level of MMP9 m⁶A methylation in shMETTL3 and shNC cells were first examined by employing the MeRIP assay. The results of the m⁶A quantification assays implied that the MMP9 mRNA m⁶A modification levels were higher in HCT116 shNC cells (Fig. 3A) and SW480 shNC cells (Fig. 3B) compared with shMETTL3 cells. Moreover, western blotting showed that the expression of MMP9 protein was downregulated in shMETTL3 cells compared with shNC cells in the two CRC cell lines (Fig. 3C). In METTL3-knockdown HCT116 and SW480 cells, the expression level of MMP9 mRNA was also significantly reduced (Fig. 3D). Next, the mechanisms behind METTL3-mediated regulation of MMP9 in CRC cells were explored by determining the protein and mRNA stability. Results from the western blotting analysis showed that the half-lives of MMP9 protein in shMETTL3 cells was comparable to that of shNC cells in both the HCT116 (Fig. 3E) and SW480 (Fig. 3F) cell lines, suggesting that decreased METTL3 expression was not related to protein stability. Given the reduced mRNA levels of MMP9 in METTL3-knockdown cells, we hypothesized that m⁶A modification might influence MMP9 mRNA stability. After treating cells with Act-D to assess mRNA abundance, the stability of mature mRNA in shMETTL3 cells was significantly lower compared with their shNC counterparts (Fig. 3G). This suggested that the mRNA stability of MMP9 was decreased in METTL3-knockdown CRC cells.

To further characterize the role of MMP9, METTL3 and MMP9 protein interactions were studied using a gain-and-loss functional experiment system. For this purpose, stable MMP9-overexpression HCT116 and SW480 cell lines were constructed and western blotting was utilized to verify MMP9 expression (Fig. 3H). The CCK-8 results showed that downregulation of METTL3 attenuated the cell viability; furthermore, overexpression of MMP9 fostered the sh-METTL3-induced downregulation of cell viability in HCT116 cells (Fig. 3I). Similar results were observed in SW480 cells, in which overexpression of MMP9 restored the shMETTL3-induced downregulation of cell viability (Fig. 3J). Additionally, the Transwell assay result showed that the MMP9 overexpression could prevent the inhibition of cell invasion caused by METTL3-knockdown (Fig. 3K). Therefore, the results indicated that METTL3 facilitates CRC tumorigenesis by enhancing the expression of MMP9 through m⁶A modification and downregulating the decay rate of MMP9 mRNA.

Stable METTL3-overexpression HCT116 cells were constructed and western blotting was utilized to verify METTL3 expression (Fig. 4A). To investigate the role of METTL3 in vivo, a subcutaneous xenotransplantation model was conducted to evaluate its contribution to CRC progression. BALB/c nude mice were subcutaneously injected with HCT116 cells with either upregulated or downregulated METTL3 expression, thereby establishing CRC xenograft models (Fig. 4B). After 24 days, the mice were sacrificed and the tumor tissues were excised, weighed and imaged (Fig. 4C). The tumors from the METTL3 overexpression cells grew more rapidly and the tumor weights were heavier compared with the control cells, whereas knockdown of METTL3 significantly repressed tumor growth compared with the control cells (Fig. 4D). The tumors derived from cells with METTL3 overexpression exhibited significantly elevated METTL3 and MMP9 mRNA levels compared with the control group. Conversely, tumors from the METTL3 knockdown cells showed a significant reduction in METTL3 and MMP9 mRNA levels relative to the control group (Fig. 4E). The IHC results showed that the shMETTL3 HCT116 ×enograft exhibited lower expression levels of METTL3 and MMP9 than the control xenografts, while METTL3-overexpression HCT116 ×enograft exhibited higher expression levels of METTL3 and MMP9 (Fig. 4F). In summary, the findings indicated that the m⁶A modification facilitated by METTL3 promoted CRC development by upregulating MMP9 expression in vivo.