Circ-ZBTB38 as an oncogenic circular RNA: Mechanisms in disrupting RalGAP complexes and its clinical value in melanoma

Abstract

Objective: Circular RNAs (circRNAs) hold significant potential both in the regulation of tumor progression and in clinical applications for tumors. This study aims to investigate the functional mechanism of circ-ZBTB38 in melanoma progression as well as its potential clinical application value.

Methods: Differentially expressed circRNAs in melanoma were screened from the GSE138412 dataset and validated by circBase, qPCR, and Sanger sequencing. Gain- and loss-of-function experiments were performed to assess the function of circ-ZBTB38. Molecular interactions were validated through RNA pull-down, mass spectrometry, and RIP assays; binding regions were characterized by RNAfold WebServer analysis and RNA-segment pull-down assays. Functional and regulatory mechanisms within the circ-ZBTB38/RALGAPB axis were evaluated using qPCR, ubiquitination assays, western blotting, proliferation assays, and Transwell migration/invasion assays. The potential clinical application value was explored via interfering with circ-ZBTB38 expression in mouse models and tissue microarray analysis of 80 paired specimens. The stability of circ-ZBTB38 was confirmed by RNase R digestion and actinomycin D (ActD) decay assays.

Results: We identified circ-ZBTB38 as an oncogenic circular RNA up-regulated in melanoma tissues and cell lines. Functional assays demonstrated that circ-ZBTB38 promoted melanoma cell proliferation and migration. Mechanistically, circ-ZBTB38 bound RALGAPB through its backsplicing site, accelerating its ubiquitination degradation, subsequently disrupted the catalytic subunit of RalGAPs. Clinically, high circ-ZBTB38 expression was correlated with a worse prognosis. Interfering with circ-ZBTB38 expression in vivo can significantly inhibit tumor growth and metastasis, and prolong the survival of mice.

Conclusions: We have established the circ-ZBTB38/RALGAPB axis as a novel regulatory circuit in melanoma. Circ-ZBTB38 mediates RALGAPB post-translational degradation to hyperactivate Ral signaling. This work uncovers a new mechanism of circRNA-mediated regulation of enzyme activity (RalGAPs) in signaling crosstalk and highlights circ-ZBTB38 as a prognostic biomarker and therapeutic target for melanoma.

Keywords: Melanoma; Metastasis; RALGAPB; Ral; circRNA.