#426

The molecular mechanism by which METTL3-mediated m6A methylation modification activates MAT2A to drive the invasion, migration, and cisplatin resistance of bladder cancer

ZHE ZHANG¹, Yanming Xu¹, Jianbin Bi¹

¹ First Hospital of China Medical University, Department of Urology, Shenyang

Introduction:
Bladder cancer, a common tumor in the urinary system, is treated with transurethral resection and platinum-based chemotherapy. While some patients achieve a cure, recurrence due to chemoresistance leads to low five-year survival rates, making it essential to understand cisplatin resistance mechanisms.
N6-methyladenosine (m6A) is the main RNA modification in eukaryotes, influencing mRNA translation and stability. In bladder cancer, METTL3 acts as a "writer" for m6A, but its role in cisplatin resistance is unclear.
Methionine adenosyltransferase 2A (MAT2A) is vital for methionine metabolism, converting ATP and L-methionine into S-adenosylmethionine (SAM). Investigating MAT2A's role in bladder cancer and its connection to cisplatin resistance is necessary.
This study will examine how METTL3 regulates MAT2A via m6A methylation and its impact on bladder cancer invasion, migration, and cisplatin resistance.

Materials and Methods:
1. Cell Model and Transcriptome Analysis: The cisplatin-resistant bladder cancer cell line UM-UC3 will be established to determine IC50 values using CCK8 assays. RT-qPCR will detect resistant gene expressions, and clinical samples will be evaluated for MAT2A expression through immunohistochemistry and Western blotting.
2. Functional Assays: MAT2A will be knocked down in bladder cancer cells, followed by transwell assays to analyze migration and invasion. CCK8 assays and flow cytometry will assess sensitivity to cisplatin. Tumor volumes and weights will be measured in mouse models.
3. METTL3 Investigation: METTL3 expression in bladder cancer will be analyzed using the GEPIA database. Paired clinical tissue samples will undergo RT-qPCR and Western blot analysis, exploring the effects of METTL3 knockdown on cell behavior and cisplatin sensitivity.
4.Molecular Mechanism Exploration: The influence of METTL3 knockdown on MAT2A expression will be analyzed. RNA pulldown assays will test the interaction between METTL3 and MAT2A mRNA, and m6A methylation sites on MAT2A will be validated.

Results:
1. Cisplatin-resistant cells show reduced sensitivity to cisplatin, with upregulation of genes like MDR1 and MGMT. Transcriptomic analysis indicates increased MAT2A expression, confirmed by Western blot.
2. METTL3 is significantly overexpressed in bladder cancer tissues, correlating positively with MAT2A. Knockdown of METTL3 inhibits cell migration, invasion, and enhances cisplatin sensitivity.
3. METTL3 directly binds to MAT2A mRNA, reducing its stability and regulating expression. MeRIP experiments confirm methylation sites on MAT2A mRNA, with dual-luciferase assays showing that METTL3 promotes m6A methylation.