N6-methyladenosine methyltransferase METTL3 promotes colorectal cancer cell proliferation through enhancing MYC expression

Cell culture and clinical samples

Human CRC cell lines SW620, HCT116, SW480, HT-29, Lovo, Caco-2 and the human embryonic kidney cell line 293T (HEK-293T) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). All the cells were cultured in Dulbecco modifed Eagle medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China), 1% penicillin and streptomycin (Hyclone, Logan, UT, USA). Tissue samples were collected from the First Affiliated Hospital of Chongqing Medical University. The criteria for patients enrolled in the study were as follows: pathological diagnosis confirmed as colorectal cancer, no familial genetic syndrome (such as Lynch syndrome); no radiotherapy or chemotherapy prior to surgery.

The RNAseq gene expression (HiSeqV2, Illumina, San Diego, CA, USA) data containing 434 CRC samples was downloaded from TCGA hub, UCSC Xena (https://xenabrowser.net/datapages/) and then analyzed using the R package.

Establishment of stable knockdown and overexpression cell lines

For knockdown of METTL3, the shRNA expression vectors were generated by annealing two complementary shRNA oligonucleotide strands synthesized by TsingKe (Chongqing, China) and then inserting into the pGreen-Puro vector (GenePharma, shanghai, China). The target sequences of shRNA are listed in Table S1. For overexpression of METTL3, the cDNA sequence containing METTL3 ORF was synthesized by TsingKe (Chongqing, China) and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-CopGFP-T2A-puro at EcoRI and BamHI sites. The plasmid encoding human MYC was obtained from GeneChem (Shanghai, China). Plasmids were transfected into cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. For the generation of shRNA and overexpression plasmid lentivirus, HEK-293T cells were cotransfected with pGreen-Puro shRNA vectors or overexpression plasmid and packaging plasmids, psPAX2 and pMD2.G (Addgene, Cambridge, MA, USA). The virus supernatant was collected after 48 or 72 hours and then mixed with 5 μg/mL polybrene and directly added to HCT116 or SW620 cells for infection. The cells were then selected using 1 µg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) for 5 days. The knockdown and overexpression efficiency of stable cell lines were analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting.

Reverse transcription-quantitative PCR (RT-qPCR)

RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Reverse transcription PCR and quantitative real-time PCR were performed using a PrimeScript RT kit (Takara, Dalian, China) and SYBR green I Master Mix (Bimake, Houston, TX, USA). PCR amplification reactions were performed in triplicates for each cDNA sample. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control, and all gene-specific primers used in qPCR analysis are listed in Table S1.

Immunohistochemistry (IHC) staining

The CRC specimens were collected and immediately fixed with 10% paraformaldehyde. The fixed tissues were embedded in paraffin and then sectioned for subsequent experiments. The paraffin sections were incubated at 55°C for 4 hours, then continuously soaked in fresh xylene solution for 20 minutes twice to remove paraffin. Sections were hydrated with 100%, 95%, 85%, and 70% ethanol and deionized water, respectively, then immersed in citrate buffer solution and heated 20 minutes for antigen retrieval. After washing three times with phosphate-buffered saline (PBS) solution, sections were incubated with 0.5% Triton X-100 for 30 minutes. IHC was then performed with a biotin-streptavidin horseradish peroxidase (HRP) detection kit (ZSGB, Beijing, China) according to the manufacturer’s instructions. Briefly, sections were treated with 3% H₂O₂ for 8 minutes, blocked with 3% bovine serum albumin for 1 hour, and then incubated with primary antibody targeting METTL3 [1:300, ab195352; Abcam, Cambridge, UK, MYC (1:200), ab32072; Abcam] overnight at 4°C. The above sections were incubated with HRP-conjugated secondary antibody for 30 minutes at room temperature, developed in diaminobenzidine (brown) solution for 3-5 minutes, and then counterstained with hematoxylin (blue). Images were acquired in multiples at 100 × or 400 × using a Leica inverted microscope (Leica, Wetzlar, Germany).

Western blot analysis

Protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitors at 100:1 (Beijing Dingguo, Beijing, China). Equal amounts of protein (30-50 μg) were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were then blocked with 5% skim milk-TBST for 1.5 hours at room temperature and incubated with the primary antibody overnight at 4°C. The corresponding primary antibodies used for western blot analysis were as follows: METTL3 (1:1,000; ab195352; Abcam), MYC (1:1,000; ab32072; Abcam), and GAPDH (1:5,000; 10494-1-AP; Proteintech, Wuhan, China). After incubating with HRP-conjugated anti-rabbit IgG secondary antibody (1:5,000; Proteintech), the protein signals were developed with an ECL chemiluminescence solution (Advansta, San Jose, CA, USA).

Cell cycle and apoptosis analysis, colony formation assay, CCK-8 assay

Cell cycle and apoptosis analysis were performed using flow cytometry by Academy of Life Sciences (Chongqing Medical University, Chongqing, China). For the colony formation assay, 1,000 HCT116 cells or 2,000 SW620 cells were seeded in 6-well plates. After culturing for 10-12 days, cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% Crystal Violet for 10 minutes. After washing three times with PBS and drying at room temperature, the numbers of colonies containing more than 50 cells were counted. Cell proliferation was further detected by using a CCK-8 kit (MCE, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions. Briefly, HCT116 or SW620 cells were seeded into 96-well plates at 2,000 cells/well as triplicates and incubated at 37°C in a humidified atmosphere containing 5% CO₂. After the cells were attached, 10 μL CCK-8 reagents were added to each well of the plate at the indicated times. After 2 hours of incubation, the absorbance at 450 nm of each well was measured using a microplate reader.

Migration and invasion assay

For cell migration and invasion Transwell assays, 5 × 10⁴ HCT116 cells or 1 × 10⁵ SW620 cells in 200 μL serum-free medium were plated on the top chambers of non-coated membranes (pore size, 8 μm; Corning, NY, USA) for the migration assay and Matrigel-coated membranes (BD Biosciences, San Jose, CA, USA) for the invasion assay, while 500 μL culture media with 10% FBS was added to the bottom. After 24-48 hours incubation, the cells in the top chambers were carefully removed by a cotton swab. The cells migrating to the lower surface of the membrane were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% Crystal Violet for 10 minutes. The migrated and invaded cells were counted with a microscope (200 ×).

Measurement of total m6A levels

Total RNA m6A level was measured using a colorimetric ELISA assay with the m6A RNA methylation quantification kit (P-9005; Epigentek, Farmingdale, NY, USA) following the manufacturer’s instructions. Measurements were conducted in triplicates for each RNA sample. A total of 200 ng RNA was used per well.

RNA immunoprecipitation (RIP)

RNA Immunoprecipitation (RIP) was performed according to a previous report [25] with some changes. Briefly, 10⁷ cells were collected by trypsinization, then lysed in 1 mL RIP buffer [(50 mM Tris, pH 7.4), 150 mM NaCl, and 1% NP-40, supplemented with 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 100 U/mL RNase inhibitor (Beyotime)] on ice for 10 minutes and then centrifuged at 16,000 × g for 15 minutes in a microcentrifuge at 4°C. Two micrograms anti-IGF2BP1 antibody (8482S; Cell Signaling Technology, Danvers, MA, USA) or normal rabbit IgG antibody (B900610; Proteintech) were incubated with 1 mL cell lysates at 4°C overnight with gentle rotation. After incubation, 50 μL precleared protein A/G beads (B23202; Bimake, Houston, TX, USA) were added to the above complexes and incubated for 4 hours at 4°C with gentle rotation. The beads were collected by a magnetic separator and washed three times in 500 μL RIP buffer. Next, the beads were resuspended in 1 mL of TRIzol and subjected to RNA extraction. Then, RT-qPCR was used to analyze the mRNA expression levels of corresponding genes using immunoprecipitated samples and input samples. The RNA levels in the immunoprecipitated samples were normalized to the input samples.

Tumorigenesis in nude mice

BALB/c male nude mice (4-6 weeks old) were purchased from the Animal Experimental Laboratory of Chongqing Medical University and housed under special pathogen-free condition. METTL3 stable knockdown or overexpression HCT116 cells were collected and resuspended at a density of 5 × 10⁶ or 3 × 10⁶ cells per 150 μL PBS, respectively. The randomly grouped mice (five in each group) were then injected subcutaneously into two flanks with 150 μL of the cell suspensions within 1 hour after cell collection. From the 4th day after injection, tumors were measured every 4 days using a vernier caliper, and the tumor volume was calculated by the following formula: volume (mm3) = 0.5 × length × width². After 24 days, the mice were euthanized and the tumors were isolated, photographed, and weighed.

Statistical analysis

GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Data are presented as the mean ± SD. Two-tailed Student’s t-test was used to compare differences between groups. Pearson’s correlation test was used to measure the strength of association between two variables. The chi-square test was used to assess correlations with clinical pathologies. P-value < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Upregulation of METTL3 was associated with clinicopathological features in CRC

To investigate the correlation between the expression of METTL3 and clinicopathological features in CRC, we first detected the expression of METTL3 mRNA in 20 pairs of CRC tumor tissues and adjacent normal tissues by RT-qPCR, and found that compared with adjacent tissues, the expression level of METTL3 mRNA was significantly upregulated in CRC tissues (Figure S1A). Next, IHC staining was used to detect the protein level of METTL3 in 45 CRC samples. The results showed that METTL3 was significantly upregulated in CRC, and predominantly localized in the nucleus of CRC cells (Figure 1A and 1B). Additionally, we also observed a negative correlation between the expression level of METTL3 and the differentiation status of CRC tissues (Figure 1A and 1C). Western blot analysis of 40 CRC samples from the cohort also showed that the expression level of METTL3 in CRC tumor tissues was significantly higher than that in adjacent normal tissues (Figure 1D). All these data showed that METTL3 was frequently upregulated in CRC. In addition, compared with other CRC cell lines, the expression of METTL3 was higher in HCT116 and SW620 cells (Figure 1E and 1F).

To further explore the clinical relevance of METTL3 in CRC, we divided the 45 CRC samples into two groups according to METTL3 IHC staining levels: the METTL3 positive expression group (n = 19, 42.2%) and the negative expression group (n = 26, 57.8%) (Figure S1B). By analyzing the correlation between the expression of METTL3 and clinicopathological features in 45 CRC patients, we found that METTL3 expression was significantly associated with tumor size (P = 0.041), stages (P = 0.019), and the degree of tumor differentiation (P = 0.040) (Table 1). Therefore, we concluded that METTL3 was frequently upregulated in human CRC and might facilitate the development of CRC by promoting cell proliferation and inhibiting differentiation.

METTL3 knockdown inhibited CRC cell proliferation in vitro

To investigate the function of METTL3 in CRC, we established stable METTL3 knockdown HCT116 and SW620 cell lines with two different shRNA sequences (shMETTL3#1 and #2). Both mRNA and protein level analysis indicated that METTL3 was successfully knocked down (Figure 2A). The CCK-8 assay was used to examine the effect of METTL3 on cell proliferation. The results showed that METTL3 knockdown significantly inhibited cell proliferation, both in HCT116 and SW620 cells (Figure 2B). Colony formation assays were conducted to further evaluate the effect of METTL3 on cell proliferation. The results showed that METTL3 knockdown suppressed cell colony formation (Figure 2C). In addition, the cell cycle was analyzed by flow cytometry. The results showed that knockdown of METTL3 inhibited G0/G1 to S cell cycle progression (Figure 2D). Apoptosis analysis showed no significant changes in both stable knockdown HCT116 and SW620 cells (Figure S2A and S2B). Furthermore, Transwell assays were performed to test the effect of METTL3 on cell migration and invasion. The results showed that knockdown of METTL3 significantly suppressed cell migration and invasion in HCT116 cells but had no significant effect on SW620 cells (Figure S2C and S2D). Thus, whether METTL3 could affect the migration and invasion ability of colorectal cancer cells remains to be further confirmed. Taken together, these results indicated that METTL3 knockdown inhibited CRC cell proliferation by suppressing the cell cycle G0/G1 to S phase transition.

Overexpression of METTL3 promoted CRC cell proliferation in vitro

To further characterize the role of METTL3 in CRC, we constructed stable cell lines overexpressing METTL3 in HCT116 and SW620 cells. Overexpression of METTL3 was verified at both mRNA and protein levels (Figure 3A). The CCK-8 assay was performed to test the effect of METTL3 overexpression on cell proliferation. Our results showed that overexpression of METTL3 significantly promoted cell proliferation compared with the empty vector control (Figure 3B). Consistently, the colony formation assay also demonstrated that overexpression of METTL3 promoted CRC cell proliferation (Figure 3C). Additionally, cell cycle analysis also showed that upregulation of METTL3 accelerated the G0/G1 to S phase transition both in HCT116 and SW620 cells (Figure 3D). Taken together, these results showed that METTL3 overexpression promoted CRC cell proliferation by accelerating the cell cycle G0/G1 to S phase transition.

METTL3 promoted MYC expression partially via an m6A-IGF2BP1 dependent manner in CRC cells

To identify METTL3 downstream targets in CRC, RT-qPCR was performed to detect the expression change of genes associated with cell proliferation in METTL3 stable knockdown cells, such as IGF2, EGFR, CTNNB1, P21, P16, CDK1, CDK2, CDK4, CDK6, CCND1, CDC25A, GADD45A, and MYC. The results showed that no other genes than MYC among these genes represented consistent and significant downregulation of both HCT116 and SW620 METTL3 in the stable knockdown cells (Figures 4A and S3A). Next, we examined the expression change in protein level of MYC by western blot analysis and found that MYC also was downregulated in both HCT116 and SW620 METTL3 stable knockdown cells compared with their controls (Figure 4B). To further study the relationship between METTL3 and MYC, RT-qPCR and western blot analysis were performed to detect mRNA and protein levels of METTL3 and MYC in six CRC cell lines, respectively. The result showed that there was a significantly positive correlation between METTL3 and MYC for both mRNA and protein expression levels in the six CRC cell lines (Figure 4C and 4D). Additionally, we analyzed the correlation of METTL3 and MYC expression levels using RNA-seq gene expression data from TCGA, which contained 434 CRC samples. The results showed that the expression of MYC mRNA was also significantly positively correlated with the expression of METTL3 (Figure S3B). These results suggested that METTL3 enhanced the expression of MYC in CRC cells. To further verify the modulatory role of METTL3 on MYC, we performed MYC rescue experiments and found that overexpression of MYC partially recovered the proliferation ability in METTL3 stable knockdown HCT116 and SW620 cells (Figures 4E and S3C), which further indicated that MYC was a downstream target gene regulated by METTL3 in CRC.

The human MYC gene is an oncogene, encoding a nuclear phosphoprotein that plays an essential role in cell cycle progression, apoptosis, and cellular transformation, which is a well-known target of IGF2BP1 [26,27]. A previous study reported that m6A modifications were enriched in a coding region instability determinant of MYC, which could be recognized by IGF2BP proteins and enhanced its mRNA stability and translation. To investigate whether METTL3 regulated the expression of MYC in the manner of m6A-IGF2BP1-dependent in colorectal cancer, we examined the total RNA m6A modification level changes in both METTL3 stable knockdown HCT116 and SW620 cells using m6A colorimetric ELISA assays. The result showed that the total RNA m6A levels were significantly decreased upon knockdown of METTL3 both in HCT116 and SW620 cells (Figure 4F). Furthermore, IGF2BP1-RIP experiments were performed in METTL3 stable knockdown HCT116 cells and its corresponding controls. The results showed that MYC mRNA binding to IGF2BP1 significantly decreased upon METTL3 knockdown (Figure 4G), which indicated that m6A modification might facilitate the binding of IGF2BP1 to MYC mRNA. Taken together, we concluded that MYC was a downstream target of METTL3 in CRC cells, which might be regulated, at least partially in an m6A-IGF2BP1 dependent manner.

METTL3 facilitated CRC tumorigenesis by enhancing the expression of MYC in vivo

To further confirm the oncogenic role of METTL3 in CRC, subcutaneous tumor formation experiments were performed to detect the effect of METTL3 overexpression or knockdown on tumorigenesis in nude mice. During tumor formation, the volume of the tumor was measured every 4 days from the 4th day after injection. Mice were euthanized at the 24th day and the tumors were removed, photographed, and weighed (Figure 5A and 5C). The results showed that tumors generated by METTL3 overexpression cells were significantly larger and heavier than controls (Figure 5B). In contrast, the METTL3 knockdown group showed a significant decrease of tumor size and weight compared with its control group (Figure 5D). These results indicated that METTL3 facilitated CRC tumorigenesis in vivo. To further confirm whether METTL3 promoted tumor growth in vivo by regulating the expression of MYC, proteins of the isolated tumors were extracted and subjected to western blot analysis. The results showed that overexpression of METTL3 promoted MYC expression, and coincidently, knockdown of METTL3 inhibited the expression of MYC (Figure 5E). Consistent with western blot analysis, IHC staining of mouse tumor tissues also confirmed these results (Figure 5F). Taken together, our results suggested that METTL3 promoted CRC cell proliferation by enhancing the expression of MYC in vivo.