METTL3 recruiting M2-type immunosuppressed macrophages by targeting m6A-SNAIL-CXCL2 axis to promote colorectal cancer pulmonary metastasis

Abstract

Background: The regulatory role of N6-methyladenosine (m6A) modification in the onset and progression of cancer has garnered increasing attention in recent years. However, the specific role of m6A modification in pulmonary metastasis of colorectal cancer remains unclear.

Methods: This study identified differential m6A gene expression between primary colorectal cancer and its pulmonary metastases using transcriptome sequencing and immunohistochemistry. We investigated the biological function of METTL3 gene both in vitro and in vivo using assays such as CCK-8, colony formation, wound healing, EDU, transwell, and apoptosis, along with a BALB/c nude mouse model. The regulatory mechanisms of METTL3 in colorectal cancer pulmonary metastasis were studied using methods like methylated RNA immunoprecipitation quantitative reverse transcription PCR, RNA stability analysis, luciferase reporter gene assay, Enzyme-Linked Immunosorbent Assay, and quantitative reverse transcription PCR.

Results: The study revealed high expression of METTL3 and YTHDF1 in the tumors of patients with pulmonary metastasis of colorectal cancer. METTL3 promotes epithelial-mesenchymal transition in colorectal cancer by m6A modification of SNAIL mRNA, where SNAIL enhances the secretion of CXCL2 through the NF-κB pathway. Additionally, colorectal cancer cells expressing METTL3 recruit M2-type macrophages by secreting CXCL2.

Conclusion: METTL3 facilitates pulmonary metastasis of colorectal cancer by targeting the m6A-Snail-CXCL2 axis to recruit M2-type immunosuppressive macrophages. This finding offers new research directions and potential therapeutic targets for colorectal cancer treatment.

Keywords: Colorectal cancer pulmonary metastases; Epithelial–mesenchymal transition; M2-type macrophages; METTL3; N6-Methyladenosine.

Fig. 1

METTL3 and YTHDF1 are highly expressed in CRPM. a Expression profile of WERs in COAD based on TCGA database. b Volcano plot of differential gene sequencing WERs in colorectal cancer in situ and lung metastatic cancer. c qPCR Analysis of METTL3, WTAP, METTL14, and YTHDF1 Gene Expression in colorectal In Situ Carcinoma and Pulmonary Metastatic Carcinoma. d-e Western blotting analysis of METTL3 and YTHDF1 protein expression in colorectal in situ carcinoma and pulmonary metastatic carcinoma. T: colorectal In Situ Carcinoma. M: colorectal pulmonary metastatic carcinoma. f Immunohistochemical Analysis of METTL3 and YTHDF1 Protein Expression in colorectal In Situ Carcinoma and Pulmonary Metastatic Carcinoma. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

Silencing the METTL3 gene inhibited the proliferation, invasion, and migration abilities of CRC cells while promoting apoptosis. a Representative images and quantitative analysis of CRC cell migration based on wound healing assay. b Decreased expression of METTL3 restrained CRC cell invasion ability based on transwell assay. c The colony formation ability of CRC cells silencing METTL3 or not was measured by colony formation assay. d EDU assay in CRC cells was performed the to measure the proliferation level. e CCK-8 assay was applied to measure the proliferation level of CRC cells after transfected with sh-METTL3 or not. f Flow cytometry analysis the proportion of apoptosis. g Western blotting analysis was applied to measure the protein expression level of E-cadherin, METTL3, SNAIL and Vimentin. GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Decreased expression of METTL3 inhibits tumor growth in vivo. a Comparison of subcutaneous tumor size in nude mice after injecting RKO/SW480 CRC cells stably transfected with Sh-NC/Sh-METTL3. b The tail vein-lung metastasis model. Cells were injected into the tail vein to produce tumor cell lung metastasis. c-d IHC analysis of METTL3, SNAIL and Vimentin for tissues of subcutaneous tumor and lung metastasis tumor. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

METTL3 regulates tumor process via Snail. a Western blotting analysis was applied to measure the protein expression level of E-cadherin, METTL3, SNAIL and Vimentin. GAPDH was used as a loading control. b Representative images and quantitative analysis of CRC cell migration based on wound healing assay. c Increased expression of SNAIL in sh-METTL3 CRC cell promoted CRC cell invasion ability based on transwell assay. d The colony formation ability of CRC cells overexpressing SNAIL or not was measured by colony formation assay in sh-METTL3 CRC cell. e EDU assay in CRC cells was performed the to measure the proliferation level. f CCK-8 assay was applied to measure the proliferation level of CRC cells after transfected with ov-SNAIL or not in sh-METTL3 CRC cell. g Flow cytometry analysis the proportion of apoptosis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

METTL3 regulates tumor EMT process via m6A-Snail. a The MeRIP qRT-PCR analysis of SNAIL mRNA in sh-NC and sh-METTL3 CRC cells. b Precursor and mature mRNA of SNAI1 in sh-NC and sh-METTL3 CRC cells. c The relative levels of the nuclear versus cytoplasmic SNAI1 mRNA in sh-NC and sh-METTL3 CRC cells. d sh-NC and sh-METTL3 CRC cells were pretreated with Act-D for 90 min, then precursor (right) or mature (left) SNAIL mRNA were analyzed at indicated times. e sh-NC and sh-YTHDF2 CRC cells were pretreated with Act-D for 90 min, then mature SNAI1 mRNA were analyzed at indicated times. f Western blotting analysis was applied to measure the protein expression level of SNAIL and YTHDF1. GAPDH was used as a loading control. g GSEA analysis of SNAIL high-expression and low-expression groups utilizing TCGA COAD data. h Western blotting analysis was applied to measure the protein expression level of SNAIL, P65 and P-P65. Tubulin was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

METTL3-expressing CRC Cells Recruits M2-type macrophage Via Secreting CXCL2. a Chromatin immunoprecipitation sequence data show that Snail might directly bind to Cxcl2 proximal promoters. b (Left) Schematic representation of CXCL2 promoter organization, and the luciferase reporter constructs pGL-CXCL2 (1606 bp: − 1606 to + 104 bp, 942 bp: − 942 to + 104 bp, 457 bp: − 457 to + 104 bp). TSS: transcriptional start site, E1: exon1, and Luc: luciferase. The green bars indicate E-boxes (CANNTG), which are the binding sites of Snail. (Right) Relative luciferase activities are shown. c ELISA to analyze the expression of CXCL2 in sh-NC/sh-SNAIL RKO cells (left), and the expression of CXCL2 in sh-NC/sh-SNAIL RKO cells (right), treated with or without BAY11-7082 (NF-κB inhibitor) at 10 μM for 24 h. d M2-type macrophage were seeded in the top chamber of the transwell containing 100 mL 1640 medium with or without CXCR2 inhibitor (SB265610, 10 mM). On the other hand, the bottom chamber contained 600 mL of CRC cell conditioned medium (no fetal bovine serum) with or without recombinant CXCL2 protein (1 ng/mL). After 4-h incubation, cells that have completely migrated to the bottom chamber were counted. e Immunofluorescence analysis of CD163 protein expression in colorectal in nude mice specimen. f Immunofluorescence analysis of CD163 protein expression in colorectal in situ carcinoma and pulmonary metastatic carcinoma. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001