Deciphering the mechanisms of transcriptome remodeling in residual HCC recurrence post-radiofrequency ablation via long-read sequencing

Abstract Background Insufficient radiofrequency ablation (IRFA) remains a major clinical challenge in hepatocellular carcinoma (HCC) treatment, often leading to rapid tumor recurrence. However, the full-length transcriptome landscape and the role of alternative polyadenylation (APA) in post-IRFA recurrence remain poorly understood. Methods We performed Oxford Nanopore Technologies long-read RNA sequencing (ONT-lrRNA-seq) on paired HCC and adjacent non-tumor tissues from five patients, categorized into four groups: pre-RFA tumor, pre-RFA adjacent, post-RFA tumor, and post-RFA adjacent tissues. Differential gene expression, transcript usage (DTU), and APA events were systematically analyzed. Results Our findings demonstrate that residual HCC cells adopt a therapy-resistant state through the synergistic regulation of gene expression, transcript usage, and polyadenylation. This is characterized by the persistent overexpression of proliferation and anti-apoptosis drivers (e.g., CDK1, BIRC5), accompanied by specific DTU events in DNA repair mechanisms (e.g., RHNO1, ANAPC13). Furthermore, IRFA significantly reshaped the APA landscape, with key oncogenes (e.g., SAE1, SPINK1) exhibiting APA-mediated 3’UTR alterations that may enhance transcript stability and potentially facilitate miRNA escape. Collectively, these events constitute the molecular basis for the survival and proliferation of residual HCC cells. Conclusion This study demonstrates that IRFA induces a state of molecular adaptation characterized by the synergistic regulation of gene expression, isoform switching, and APA. These mechanisms collectively promote the survival and proliferation of residual HCC cells, providing novel insights and identifying candidate targets that warrant further functional investigation for preventing post-ablation recurrence.