CRISPR-mediated MLH1 disruption suppresses endometrial cancer growth via genomic instability induction and
Baoling Xing1, Xiaoying Zhang2, Li Shen2
1Department of Pathology, Affiliated Zhoupu Hospital of Shanghai University of Medicine and Health Sciences, Shanghai, China. 793506861@qq.com.
Introduction:
MutL homolog 1 (MLH1) loss is a defining molecular feature of endometrial cancer (EC) and a principal driver of microsatellite instability (MSI). Ishikawa cells harbor intrinsic MLH1 promoter hypermethylation, resulting in reduced but not abolished MLH1 expression and placing these cells in a vulnerable, partially compromised mismatch repair state. This study explores the effects of MLH1 knockdown (MLH1-KD) on MSI, cellular functions, signaling pathways, and tumor growth in Ishikawa EC cells.
Material And Methods:
Using CRISPR/Cas9, we created an MLH1-KD Ishikawa EC cell line, validated through Sanger sequencing, qRT-PCR, western blotting, comet assays, and γ-H2AX analysis. Functional assays assessed proliferation, migration, and cell cycle progression and apoptosis. RNA sequencing identified global transcriptomic changes, and Wnt/β-catenin pathway activity was measured by a dual-luciferase reporter assay. A xenograft model evaluated tumor growth in vivo.
Results:
MLH1-KD cells showed MSI-H characteristics, increased DNA damage, and downregulation of key EC-related genes. Functionally, MLH1-KD led to significant reductions in cell proliferation and migration, which was accompanied by cell cycle arrest and a marked increase in apoptosis. RNA sequencing revealed profound alterations in the Wnt signaling pathway. Crucially, this was confirmed by a dual-luciferase reporter assay, which showed a significant inhibition of Wnt/β-catenin signaling activity. In vivo, MLH1-KD significantly decreased tumor weight and size in nude mice.
Conclusions:
In EC cells with pre-existing MLH1 promoter methylation, MLH1-KD leads to MSI-H, enhances genomic instability, disrupts Wnt signaling, impairs cellular functions, and inhibits tumor growth, highlighting Wnt signaling and MSI-H as potential therapeutic targets in EC.
