METTL16 Promotes Stability of SYNPO2L mRNA and leading to Cancer Cell Lung Metastasis by Secretion of COL10A1 and attract the Cancer-Associated Fibroblasts

Abstract

The occurrence of metastasis is a major factor contributing to poor prognosis in colorectal cancer. Different stages of the disease play a crucial role in distant metastasis. Furthermore, m6A has been demonstrated to play a significant role in regulating tumor metastasis. Therefore, we conducted an analysis of transcriptome data from high-stage and low-stage colorectal cancer patients in The Cancer Genome Atlas (TCGA) to identify genes associated with m6A-related regulation. We identified SYNPO2L as a core gene regulated by m6A, and it is correlated with adverse prognosis and metastasis in patients. Additionally, we demonstrated that the m6A writer gene Mettl16 can regulate the stability of SYNPO2L through interaction with YTHDC1. Subsequently, using Weighted Gene Co-expression Network Analysis (WGCNA), we discovered that SYNPO2L can regulate COL10A1, mediating the actions of Cancer-Associated Fibroblasts. SYNPO2L promotes the secretion of COL10A1 and the infiltration of tumor-associated fibroblasts, thereby facilitating Epithelial-Mesenchymal Transition (EMT) in tumor cells and making them more prone to distant metastasis.

Keywords: Cancer-Associated Fibroblasts; Colorectal Cancer; Epithelial-Mesenchymal Transition; Metastasis; N6-methyladenosine.

Figure 1

Analysis of Transcriptome Data from TCGA Colorectal Cancer Patients Reveals m6A-regulated Core Gene SYNPO2L as a Significant Factor in Patient Staging. (A) KM survival analysis; (B) Hallmark GSEA enrichment analysis, NES= 1.21, NOM p-val < 0.001; (C) Protein interaction network map for m6A-related genes and all associated genes; (D) Survival analysis forest plot for significant differences between m6A related genes and all associated genes; (E) Heatmap analysis of genes with significant differences between m6A-related genes and all associated genes in relation to TNM, gender, age, staging, etc. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

METTL16 Expression Significantly Correlates with The Expression of SYNPO2L, A Core Gene Related to Patient Survival Prognosis. (A) Statistical analysis of SYNPO2L mRNA expression in cancer and adjacent cancer tissue, and in late and early stage patients; (B) Statistical analysis of CST2 mRNA expression in cancer and adjacent non-tumor tissues, as well as in patients with high and low stage cancer; (C) Statistical analysis of C1QTNF9 mRNA expression in cancer and adjacent non-tumor tissues, as well as in patients with high and low stage cancer; (D) Overall survival analysis based on SYNPO2L expression levels in colorectal cancer patients from the TCGA database; (E) Overall survival analysis based on SYNPO2L expression levels in colorectal cancer patients from the TCGA database; (F) Correlation analysis of SYNPO2L with m6A-related genes; (G) qPCR analysis of METTL16 and SYNPO2L expression in CRC cancer and adjacent non-tumor tissues; (H) qPCR analysis of METTL16 and SYNPO2L expression in CRC cancer and lung metastasis; (I) qPCR analysis of the correlation between METTL16 and SYNPO2L expression. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

SYNPO2L Significantly Impacts Tumor Cell Proliferation and Migration. (A-B) EDU experiment and statistical bar graph after SYNPO2L knockdown or overexpression; (C-D) Transwell migration experiment and statistical bar graph after SYNPO2L knockdown or overexpression; (E) Subcutaneous tumor formation experiment in nude mice, comparing tumor size and weight after injecting CRC cell lines transfected with Sh-Control/SYNPO2L-Sh2/control/SYNPO2L-over, and a statistical bar graph comparing the weight of the xenograft tumors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The length of the ruler is 1cm.

Figure 4

METTL16 Promotes Tumor Proliferation and Migration by Enhancing SYNPO2L mRNA Stability Through m6A Modification. (A-B) qPCR analysis of METTL16 and SYNPO2L expression in colorectal cancer cell lines with overexpression/knockdown of METTL16; (C-D) Analysis of SYNPO2L mRNA degradation rate after METTL16 knockdown and Actinomycin D treatment; (E) EDU experiment and statistical bar graph after METTL16 knockdown or overexpression; (F) Transwell migration experiment and statistical bar graph after METTL16 knockdown or overexpression; (G) Subcutaneous tumorigenesis experiment in nude mice, comparing the size of xenograft tumors in nude mice after injection of CRC cell lines stably transfected with METTL16-Sh1/METTL16-over/control, along with a statistical bar graph comparing the weight of the xenograft tumors; (H) Immunohistochemical analysis of xenograft tumors in nude mice to detect METTL16 protein expression, and a comparative statistical bar graph of immunohistochemical scoring. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The length of the ruler is 1cm.

Figure 5

YTHDC1 Recognizes and Promotes SYNPO2L mRNA Stability Through METTL16-Mediated m6A Modification. (A) MeRIP-qPCR assessment of SYNPO2L m6A levels and statistical bar graph analysis after METTL16 knockdown;(B) TCGA database analysis of the correlation between YTHDC1 and SYNPO2L mRNA expression; (C) qPCR analysis of the correlation between YTHDC1 and SYNPO2L expression; (D-E) qPCR analysis of YTHDC1 and SYNPO2L expression in colorectal cancer cell lines with overexpression/knockdown of YTHDC1; (F-G) Analysis of SYNPO2L mRNA degradation rate after YTHDC1 knockdown/overexpression and Actinomycin D treatment; (H) Diagram of SYNPO2L gene stability regions; (I-J) Luciferase reporter gene analysis after mutating SYNPO2L gene stability regions and statistical bar graph. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

SYNPO2L Interacts with Cancer-Associated Fibroblasts (CAFs) through Secreted Proteins. (A) TCGA analysis of differentially expressed genes between high and low SYNPO2L expression groups, described in a volcano plot; (B) Protein-protein interaction network map; (C) TCGA enrichment analysis of high vs. low SYNPO2L expression groups; NES=1.61, NOM p-val < 0.0001. (D) TCGA analysis of stromal cell scoring in high vs. low SYNPO2L expression groups, shown in a bar graph; (E-F) Immunohistochemical analysis of CAFs markers and their representation in bar graphs; (G-H) Transwell cell migration assays to assess the impact of overexpressing/knocking down SYNPO2L on the migratory capabilities of colorectal cancer cells, displayed in bar graphs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, No significant.

Figure 7

SYNPO2L Influences Tumor Cell Secretion of COL10A1, Promoting Infiltration of Tumor-Associated Fibroblasts and Leading to Distant Tumor Metastasis. (A-B) Western blot analysis of COL10A1 expression levels following SYNPO2L knockdown/overexpression. GAPDH used as internal control protein; (C-D) Transwell cell migration assays evaluating the impact of knocking down/overexpressing COL10A1 on colorectal cancer cell migration, shown in bar graphs; (E-F) Transwell assays assessing the impact of COL10A1 knockdown and SYNPO2L overexpression, COL10A1 overexpression and SYNPO2L knockdown, and COL10A1 and SYNPO2L overexpression on cell migration, displayed in bar graphs; (G) Tumor formation with tail vein injection in nude mice to assess the impact of COL10A1 knockdown and SYNPO2L overexpression, COL10A1 overexpression and SYNPO2L knockdown, and COL10A1 and SYNPO2L overexpression on lung metastasis, shown in a bar graph. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 8

COL10A1 Promotes Tumor-Associated Fibroblast Infiltration Leading to EMT and Metastasis in Tumor Cells. (A) TCGA enrichment analysis of high vs. low COL10A1 expression groups; NES= 1.68, NOM p-val = 0.028. (B-C) qPCR analysis of EMT marker expression levels in colorectal cancer cells after overexpressing or knocking down COL10A1; (D-G) Subcutaneous tumor formation in MC38 cell line with COL10A1 knockdown/overexpression, followed by immunohistochemical analysis of Vimentin protein in subcutaneous grafts, shown in bar graphs; (H-I) Immunohistochemical analysis of CAFs markers in primary and lung metastatic colorectal cancer from patients, displayed in bar graphs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.