Transcriptomic analysis of zonula occludens-1 (ZO-1) knockout in ovarian cancer cell lines.

Abstract

Background: Zonula occludens-1 (ZO-1) is a crucial tight junction protein that regulates intercellular permeability and adhesion, thereby preserving the integrity of epithelial and endothelial barriers. ZO-1 is associated with tumorigenesis and the progression of epithelial-mesenchymal transition (EMT), invasion, and metastasis. In our previous study, knockout (KO) of ZO-1 using clustered regularly interspaced short palindromic repeats (CRISPR) reduced proliferation but increased migration and invasion, suggesting that ZO-1 may have a dual role. Therefore, this study aimed to elucidate the role of ZO-1 in ovarian cancer by analyzing transcriptomic changes associated with ZO-1.

Objective: This study aims to elucidate the impact of ZO-1 KO on gene expression in ovarian cancer cells by performing comparative RNA sequencing (RNA-seq) analysis on two distinct ZO-1 KO ovarian cancer cell lines, SKOV3 and SNU119.

Methods: ZO-1 was knocked out in SKOV3 and SNU119 cells using CRISPR-Cas9 technology. After identifying differentially expressed genes (DEGs) through RNA sequencing, Gene Ontology (GO) and pathway enrichment analyses were performed. The selected targets were subsequently validated using reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis to assess both transcript- and protein-level expression changes.

Results: Transcriptomic analysis revealed over 400 DEGs in each cell line. Of these, 14 genes were consistently upregulated in both cell lines, while 24 genes were consistently downregulated. The common DEGs were visualized using a heatmap, and a subset of these genes was further validated by RT-qPCR and Western blot analyses. TGFB2 expression was consistently altered at both the mRNA and protein levels following ZO-1 KO in both cell lines. Similar expression patterns were observed for THBS1, VCAN, ITGB8, SEMA3A, and GAS6. The concordant changes observed in transcriptomic and protein analyses suggest a consistent association between ZO-1 KO and TGFB2 expression.

Conclusion: ZO-1 KO in ovarian cancer cells induces substantial transcriptional reprogramming, particularly affecting genes associated with extracellular matrix organization and signaling pathways. Multiple candidate genes showed consistent alterations at both the mRNA and protein levels, supporting the robustness of the observed transcriptional changes. These findings provide a framework for understanding ZO-1-associated regulatory networks in ovarian cancer.