M6A modification has been proved to play an important role in many biological processes. METTL3 as the main methyltransferase for methylation process has been found to be upregulated in many cancers, including CRC. Here, we investigate m6A modification and the underlying mechanism of METTL3 in the development of CRC. The expression of METTL3 was detected in large clinical patient samples. To evaluate the function of METTL3 in vitro and in vivo, colony formation, CCK-8, cell migration and invasion assays were performed. To find out the downstream target of METTL3, GEO dataset was re-mined. We analyzed expression and metastasis-related miRNA by Pearson correlation, and miR-1246 was selected. Here, to identify the downstream target of miR-1246, Targetscan and miRWalk were used. RIP and luciferase reporter assay further confirmed SPRED2 as the direct target of miR-1246. We found that upregulated METTL3 is responsible for abnormal m6A modification in CRC and correlates positively with tumor metastasis. The gain- and loss-of-function indicates that METTL3 promotes cell migration and invasion in vitro and in vivo. Additionally, we confirmed that METTL3 can methylate pri-miR-1246, which further promotes the maturation of pri-miR-1246. By using bioinformatics tools, anti-oncogene SPRED2 was identified as the downstream target of miR-1246, wherein downregulated SPRED2 further reverses the inhibition of the MAPK pathway. The present study demonstrates that the METTL3/miR-1246/SPRED2 axis plays an important role in tumor metastasis and provides a new m6A modification pattern in CRC development.

Background: Early-onset colorectal cancer (EOCRC) is associated with a poorer prognosis relative to late-onset colorectal cancer (LOCRC), and its incidence has witnessed a gradual escalation in recent years. This necessitates a comprehensive examination of the underlying pathogenesis and the identification of therapeutic targets specific to EOCRC patients. The present study aimed to delineate the distinct molecular landscape of EOCRC by juxtaposing it with that of LOCRC. Methods: A total of 11 344 colorectal cancer patients, diagnosed between 2003 and 2022, were enrolled in this study, comprising 578 EOCRC cases and 10 766 LOCRC cases. Next-generation sequencing technology was employed to assess the tumor-related mutation and tumor mutation burden (TMB) in these patients. PD-L1 expression was quantified using immunohistochemistry. Microsatellite instability (MSI) was determined via capillary electrophoresis (2B3D NCI Panel). Results: Upon comparing LOCRC with EOCRC patients, the latter group demonstrated a tendency towards advanced TNM stage, lower tumor differentiation, and less favorable histological types. Among LOCRC patients, those with MSI-H status were found to have an earlier TNM stage compared to those with MSI-L/MSS status. Significantly, the incidence of MSI-H was notably higher in EOCRC (10.2%) compared to LOCRC (2.2%). Mutations in the 7-gene panel (ARID1A, FANCI, CASP8, DGFRA, DPYD, TSHR, and PRKCI) were more prevalent in LOCRC. Within the EOCRC cohort, patients with the MSI-H subtype displayed an earlier TNM stage but concurrently exhibited poorer tissue differentiation and a higher frequency of mucinous adenocarcinoma. Among EOCRC patients, FBXW7, FAT1, ATM, ARID1A, and KMT2B mutations were significantly enriched in the MSI-H subgroup. A comparative analysis of MSI-H patients revealed heightened mutation frequencies of FGFBR2, PBRM1, RNF43, LRP1B, FBXW7, ATM, and ARID1A in the EOCRC group. Furthermore, EOCRC patients demonstrated a higher overall TMB, particularly in the MSI-H subtype. PD-L1 expression was elevated in EOCRC and positively associated with MSI status. Conclusions: This study revealed a significantly higher MSI-H distribution rate in EOCRC, and EOCRC exhibits a distinct mutational signature coupled with higher PD-L1 expression. These findings hold promise in guiding personalized therapeutic strategies for improved disease management in EOCRC patients.

Cell division cycle associated 2 (CDCA2), upregulated in lung adenocarcinoma and oral squamous cell carcinoma, may be related to some malignant diseases. Nevertheless, its role in colorectal cancer (CRC) remains unknown. CDCA2 expression was analyzed using The Cancer Genome Atlas (TCGA), quantitative real-time PCR (qRT-PCR), and immunohistochemistry. The impact of CDCA2 on cell proliferation was analyzed via loss- or gain-of-function assays. Furthermore, gene set enrichment analysis was conducted to explore the potential mechanism of CDCA2 in CRC. Lastly, the expression levels of CCND1 and AKT were measured in CRC cell lines. Our study revealed that CDCA2 expression was associated with tumor progression. Through loss- or gain-of-function assays, we found that upregulation of CDCA2 promoted the proliferation of DLD-1 cells, however, downregulation of CDCA2 in SW480 cells restrained proliferative capacity both in vitro and in vivo. The results of flow cytometry showed that CDCA2 promoted cell cycle progression via upregulation of CCND1 in CRC cell lines. In the following experiments, we found that CDCA2 regulated CCND1 expression through activating the PI3K/AKT pathway, and confirmed this using a specific PI3K inhibitor (LY294002). This study demonstrates that overexpression of CDCA2 might target CCND1 to promote CRC cell proliferation and tumorigenesis through activation of the PI3K/AKT pathway.