Dataset Information

METTL3 promotes colorectal carcinoma progression by regulating the m6A-CRB3-Hippo axis.

ABSTRACT:

Background

Colorectal carcinoma (CRC) is the third most common cancer and second most common cause of cancer-related deaths worldwide. Ribonucleic acid (RNA) N6-methyladnosine (m6A) and methyltransferase-like 3 (METTL3) play key roles in cancer progression. However, the roles of m6A and METTL3 in CRC progression require further clarification.

Methods

Adenoma and CRC samples were examined to detect m6A and METTL3 levels, and tissue microarrays were performed to evaluate the association of m6A and METTL3 levels with the survival of patients with CRC. The biological functions of METTL3 were investigated through cell counting kit-8, wound healing, and transwell assays. M6A epitranscriptomic microarray, methylated RNA immunoprecipitation-qPCR, RNA stability, luciferase reporter, and RNA immunoprecipitation assays were performed to explore the mechanism of METTL3 in CRC progression.

Results

M6A and METTL3 levels were substantially elevated in CRC tissues, and patients with CRC with a high m6A or METTL3 levels exhibited shorter overall survival. METTL3 knockdown substantially inhibited the proliferation, migration, and invasion of CRC cells. An m6A epitranscriptomic microarray revealed that the cell polarity regulator Crumbs3 (CRB3) was the downstream target of METTL3. METTL3 knockdown substantially reduced the m6A level of CRB3, and inhibited the degradation of CRB3 mRNA to increase CRB3 expression. Luciferase reporter assays also showed that the transcriptional level of wild-type CRB3 significantly increased after METTL3 knockdown but not its level of variation. Knockdown of YT521-B homology domain-containing family protein 2 (YTHDF2) substantially increased CRB3 expression. RNA immunoprecipitation assays also verified the direct interaction between the YTHDF2 and CRB3 mRNA, and this direct interaction was impaired after METTL3 inhibition. In addition, CRB3 knockdown significantly promoted the proliferation, migration, and invasion of CRC cells. Mechanistically, METTL3 knockdown activated the Hippo pathway and reduced nuclear localization of Yes1-associated transcriptional regulator, and the effects were reversed by CRB3 knockdown.

Conclusions

M6A and METTL3 levels were substantially elevated in CRC tissues relative to normal tissues. Patients with CRC with high m6A or METTL3 levels exhibited shorter overall survival, and METTL3 promoted CRC progression. Mechanistically, METTL3 regulated the progression of CRC by regulating the m6A-CRB3-Hippo pathway.

SUBMITTER: Pan J

PROVIDER: S-EPMC8744223 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Colorectal cancer (CRC) is among the commonest malignant tumors of humans. Existing evidence has linked the poor prognosis of CRC with high expression of stromal antigen 3 (STAG3), but, the exact biological effect of STAG3 in CRC is still unclear. The aim of this research is to reveal the biological function and molecular mechanism of STAG3 in CRC. To investigate the differential expression of STAG3 in CRC tissues and cell lines compared to normal colon tissues and cell lines, Western blot (WB) and quantitative real-time PCR (qRT-PCR) techniques were utilized. STAG3 N6-methyladenosine (m6A) modification level were identified using m6A RNA immunoprecipitation (MeRIP). Additionally, the functional roles of methyltransferase-like protein 3 (METTL3) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in CRC were explored by manipulating their levels via knockdown or overexpression. Cell proliferation was evaluated through Cell Counting Kit 8 (CCK-8) and clone formation experiments, while cell migration was assessed through wound healing experiments. Furthermore, cell apoptosis was detected using flow cytometry, and the protein expressions associated with proliferation and apoptosis were detected using WB. To identify the specific binding of target genes, RIP and pull-down assays were employed. Finally, the biological function of STAG3 in vivo was investigated through a xenotransplantation mouse tumor model. In CRC tissues and cell lines, STAG3 was up-regulated and accompanied by m6A methylation. Additionally, the expression of METTL3 was found to be upregulated in CRC tissues. Knocking down METTL3 resulted in a decrease in both the m6A level and protein expression of STAG3, inhibited cell proliferation and migration while promoting apoptosis, which were restored through STAG3 overexpression. Furthermore, online prediction indicated the interaction between STAG3 mRNA and IGF2BP2 protein, which was further verified by RIP experiments. IGF2BP2 downregulation led to decreased STAG3 protein expression, cell proliferation, and migration, but increased apoptosis. However, these impacts were reversed by STAG3 overexpression. Finally, subcutaneous tumor experiments conducted in nude mice also confirmed that METTL3 regulated CRC progression through STAG3 in vivo. The METTL3/IGF2BP2/STAG3 axis affects CRC progression in an m6A modification-dependent manner. This may guide targeted therapy in CRC patients.

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