# CircROR1 binds HNRNPL to regulate FOXO4 pre-mRNA splicing, promoting cutaneous melanoma metastasis and serving as a therapeutic target via RNAi-loaded PEG-LNPs

## TL;DR

A circular RNA called circROR1 promotes melanoma metastasis by regulating gene splicing and can be targeted with RNAi-loaded nanoparticles for treatment.

## Contribution

Discovery of circROR1 as a novel oncogenic circular RNA and development of a targeted RNAi nanotherapy for melanoma.

## Key findings

- CircROR1 promotes melanoma metastasis by regulating FOXO4 pre-mRNA splicing through HNRNPL.
- CircROR1 upregulation increases EMT and resistance to PD-L1 antibody therapy.
- RNAi-loaded PEG-LNPs targeting circROR1 reduce tumor metastasis in vivo.

## Abstract

Circular RNAs (circRNAs) contribute to gene expression regulation by interacting with splicing factors, a process that is often disrupted in cancers such as cutaneous melanoma (CM).

A circRNA microarray analysis was performed to identify differentially expressed circRNAs. qRT‒PCR was conducted to confirm the expression of circROR1. CCK-8, colony formation, wound healing, and transwell assays were used to analyze proliferation, metastasis and apoptosis in CM cells. Xenograft models and IHC experiments were established to confirm the effects of circROR1 on tumor growth and metastasis in vivo. RNA sequencing and pull-down–MS experiments were performed to identify the mechanisms downstream of circROR1. Nuclear and cytoplasmic fractionation, along with FISH experiments, were conducted to determine the cellular localization of circROR1. To target circROR1 for CM treatment, we used a microfluidic strategy to develop FA-PEG(si-circ) nanoparticles for efficient siRNA delivery.

In CM samples, circROR1 levels were positively correlated with HNRNPL levels and tumor metastasis but negatively correlated with FOXO4 protein levels. CircROR1 was prevalent in CM, and its upregulation increased the levels of factors involved in epithelial–mesenchymal transition, cell migration, and invasion. CircROR1 overexpression conferred resistance to PD-L1-antibody therapy in CM cells by downregulating PD-L1 expression. CircROR1 recruited HNRNPL, influencing its nuclear translocation, and further prevented intron retention in FOXO4 mRNA. In HNRNPL-overexpressing CM cells, circROR1 upregulation inhibited FOXO4α expression and promoted FOXO4ζ expression. Increased FOXO4α expression counteracted circROR1’s effects and suppressed metastatic behaviors. FA-PEG(si-circ) enhanced siRNA stability and efficiency, reducing CM cell lung colonization in vivo.

This study identified circROR1 as an oncogenic circular RNA that plays a crucial role in tumor progression and metastasis. CircROR1-targeted nanotherapy is a promising option for the treatment of metastatic cancer.

The online version contains supplementary material available at 10.1186/s12943-025-02525-1.