Intranuclear paraspeckle-circular RNA TACC3 assembly forms RNA-DNA hybrids to facilitate MASH-related hepatocellular carcinoma growth in an m6A-dependent manner

Published:16 October 2025 DOI: 10.1002/cac2.70061 PMID: 41103024

Jingbo Fu, Yanping Wei, Yun Yang, Xinwei Yang, Tao Ouyang, Xianming Wang, Shuzhen Chen, Zenglin Liu, Yu Su, Jing Fu, Miao Yu, Haihua Qian, Hao Song, Shuo Xu, Ru Zhao, Xue Jiang, Yunfei Huo, Man Zhang, Pinhua Yang, Zhao Yang, Kui Wang, Liang Li, Hongyang Wang

Abstract

Background

Metabolic dysfunction-associated steatohepatitis (MASH) is anticipated to become the leading cause of hepatocellular carcinoma (HCC). Accumulating evidence indicates that N6-methyladenosine (m⁶A)-modified circular RNAs (circRNAs) play key roles in tumor malignant progression. However, the precise molecular mechanisms by which circRNAs and their m⁶A modification regulatory networks respond to metabolic reprogramming, such as lipid overload stress, to drive malignant tumor progression in the context of MASH-related HCC remain unclear. This study aimed to investigate the role and regulatory network of m⁶A-modified circRNAs in MASH-related HCC.

Methods

Epitranscriptomic microarray and in situ hybridization assays were used to validate circTACC3 expression in MASH-related HCC specimens. Palmitic acid (PA) and oleic acid (OA) was applied to NAC-organ assembled three-dimensional-organoid and HCC cell lines to imitate pathological lipid overload. The circTACC3-paraspeckle interaction was studied utilizing fluorescence lifetime imaging microscopy-Forster resonance energy transfer. An integrative analysis combining DNA-RNA immunoprecipitation combined with chromatin isolation by RNA purification (DRIP-ChIRP), γH2AX cleavage under target and tagmentation, and high-throughput/resolution chromosome conformation capture sequencing were used to study chromatin remodeling induced by circTACC3-formed RNA-DNA hybrids (R loops) at DNA double-strand break (DSB) loci during lipid overload.

Results

The most prevalent m⁶A-modified circRNA in MASH-related HCC, circTACC3, had a substantial impact on the intracellular lipid accumulation, growth, and environmental adaptive survival of tumor cells. Under lipid overload conditions, circTACC3 interacted directly with non-POU domain-containing octamer-binding protein (NONO/p54^nrb) to assemble intranuclear paraspeckle. This process was dependent on the m⁶A-modification sites of circTACC3 and facilitated its nuclear retention. Using DRIP-ChIRP-sequencing, we demonstrated that circTACC3-containing paraspeckles were recruited to DSB foci to form R loops (DSB-circTACC3-R loops). We discovered 4 highly enriched motifs of DSB-circTACC3-R loops. DSB-circTACC3-R loops further facilitated the contact and fusion of topologically associated domains (TADs) and selectively activated genes related to the malignant phenotype of MASH-related HCC. Interestingly, circTACC3-R loops exerted positive feedback control over the assembly of circTACC3 paraspeckle and clustering of TADs.

Conclusions

The m⁶A modification-dependent circTACC3-paraspeckle assembly results in the formation of R loops at DSB foci, leading to chromatin remodeling and the activation of genes involved in MASH-related HCC malignant progression. This process identifies potential therapeutic targets.