The landscape of peripheral blood RNA modifications and its clinical implications for diagnosis of hepatocellular carcinoma

While over 170 RNA modifications have been identified and implicated in various cancers, their role in hepatocellular carcinoma (HCC) progression is increasingly recognized. Despite this established relevance in tumor biology, the landscape of RNA modifications in the peripheral blood of HCC patients—and their potential diagnostic utility—remains largely unexplored. Peripheral blood samples from patients with HCC, liver cirrhosis (LC), and normal healthy (NH) controls were collected. The abundances of 55 RNA modifications were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess their diagnostic potential for HCC, particularly at early stages. Correlations among these modifications and their associations with clinical parameters were analyzed. Simultaneously, differentially expressed genes, including those encoding RNA-modifying enzymes, were screened in peripheral blood. The biological relevance of the identified signatures was subsequently validated using in vitro co-culture and in vivo syngeneic HCC mouse models. Compared to the combined non-HCC group (NH and LC), the abundances of 11 RNA modifications were significantly altered in both overall and stage I HCC groups, with N 2 , N 2 -dimethylguanosine (m 2,2 G) emerging as a key component exhibiting the most pronounced dysregulation. A diagnostic model centered on an m 2,2 G-based modification panel achieved area under the curves (AUCs) of 0.901 and 0.891 for detecting HCC and stage I HCC, respectively, demonstrating promising diagnostic potential. Notably, the incorporation of two upregulated genes in peripheral blood—IFI27 and CCR2—significantly enhanced the model’s performance, yielding improved AUCs of 0.972 and 0.968, respectively. Further analysis revealed distinct correlation patterns among RNA modifications, as well as between RNA modifications and clinical laboratory parameters, exhibiting both shared and HCC-specific features that suggest systemic reprogramming of RNA modification network in HCC. This biological relevance was confirmed by elevated m 2,2 G abundances in human lymphocytes co-cultured with HCC cells and blood from a syngeneic HCC mouse model. This study systematically profiled peripheral blood RNA modifications and provided preliminary evidence supporting their potential as diagnostic biomarkers for HCC. By integrating key modifications (m 2,2 G, m 2,2,7 G, m 6,6 A) with two mRNA markers (IFI27 and CCR2), we developed a multi-omics signature that demonstrated promising diagnostic performance, particularly for early-stage HCC.