m6A Modification Reprograms Selective RNA Cargo Loading of Extracellular Vesicles to Shape the Pre-Metastatic Niche in Hepatocellular Carcinoma
Abstract
Background and Aims: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the third leading cause of cancer-related death worldwide, with distant metastasis being the primary determinant of poor prognosis. Tumor-derived
extracellular vesicles (EVs) are critical mediators of pre-metastatic niche (PMN) formation, delivering bioactive RNA cargo to distant
stromal and immune cells. However, the molecular determinants of selective RNA loading into tumor EVs remain poorly understood. N6-
methyladenosine (m6A), the most abundant internal modification on eukaryotic messenger RNA (mRNA), regulates virtually every aspect
of RNA metabolism through writer (METTL3/METTL14/WTAP), eraser (FTO/ALKBH5), and reader (YTHDF1/2/3, IGF2BP1/2/3)
proteins. Whether m6A modification acts as a molecular barcode directing specific transcripts into EVs, and whether this process orchestrates immunosuppressive PMN remodeling in HCC, has not been systematically investigated. Method: Bulk RNA-seq datasets from The
Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) (n = 374 tumor/50 normal), International Cancer Genome Consortium
liver cancer Japan (ICGC-LIRI-JP) (n = 240 tumor/202 normal), GSE14520 (n = 225/220), and GSE76427 (n = 115/52), and single-cell
RNA sequencing (scRNA-seq) datasets from GSE149614 (10 patients, ~93, 000 cells, four tissue sites) and GSE202642 (~74, 957 cells)
were retrieved from public repositories. EV transcriptome reference data were obtained from the EVmiRNA database and Vesiclepedia.
Integrative bioinformatic analyses included differential expression profiling of 23 m6A regulatory genes, EV/cellular RNA enrichment
ratio analysis, m6A site database cross-referencing, weighted gene co-expression network analysis (WGCNA), proteinprotein interaction (PPI) network construction and hub gene analysis, cell-type deconvolution, intercellular communication inference (CellChat), and
clinical outcome evaluation. Result: Fourteen of 23 m6A regulators were significantly dysregulated in HCC across all four cohorts, with
METTL3, WTAP, YTHDF1, YTHDF2, and IGF2BP1 showing the most consistent upregulation. Cross-referencing EV-enriched transcripts
with high-confidence m6A-modified sites identified a 63-transcript m6A-marked pro-metastatic EV cargo set, including VEGFA (EV/cellular enrichment = 3.8-fold), TGF-?1 (3.2-fold), HIF1A (2.9-fold), MALAT1 (4.1-fold), and SNAI1 (2.6-fold). YTHDF2 emerged as the
principal network hub (degree centrality k = 23, betweenness centrality rank 1) through physical interactions with TSG101 and PDCD6IP/
ALIX. The YTHDF2-correlated WGCNA turquoise module (1, 241 genes; r = +0.76 with YTHDF2; r = +0.71 with vascular invasion; both
p < 0.001) was enriched in TGF-?, VEGF, ECM-receptor interaction, and epithelialmesenchymal transition (EMT) pathways. Cell-type
deconvolution confirmed that YTHDF2-high HCC was associated with M2 macrophage enrichment, regulatory T cell (Treg) accumulation,
and CD8+ T cell depletion. Single-cell analysis showed progressive YTHDF2 induction from primary tumor to portal vein tumor thrombus
(PVTT) and lymph node (LN) metastatic compartments (mean expression: 2.34 ? 2.71 ? 2.89; both p < 0.001). An m6AEV axis signature score discriminated advanced-stage HCC (TNM IIIIV) with area under the receiver operating characteristic curve (AUROC) values
of 0.84, 0.79, and 0.77 in three independent cohorts, with hazard ratios of 2.17, 1.94, and 1.82 for overall survival (OS), respectively (all p
< 0.001). Conclusion: This integrative bioinformatic study establishes m6A modification as a molecular barcode governing selective RNA
cargo loading into HCC-derived EVs, with YTHDF2 as the central sorting hub acting through physical engagement with the endosomal
sorting complexes required for transport (ESCRT) machinery. YTHDF2 and the m6AEV cargo transcripts represent mechanistically
grounded targets for therapeutic intervention and non-invasive biomarker development in advanced HCC.
extracellular vesicles (EVs) are critical mediators of pre-metastatic niche (PMN) formation, delivering bioactive RNA cargo to distant
stromal and immune cells. However, the molecular determinants of selective RNA loading into tumor EVs remain poorly understood. N6-
methyladenosine (m6A), the most abundant internal modification on eukaryotic messenger RNA (mRNA), regulates virtually every aspect
of RNA metabolism through writer (METTL3/METTL14/WTAP), eraser (FTO/ALKBH5), and reader (YTHDF1/2/3, IGF2BP1/2/3)
proteins. Whether m6A modification acts as a molecular barcode directing specific transcripts into EVs, and whether this process orchestrates immunosuppressive PMN remodeling in HCC, has not been systematically investigated. Method: Bulk RNA-seq datasets from The
Cancer Genome Atlas liver hepatocellular carcinoma (TCGA-LIHC) (n = 374 tumor/50 normal), International Cancer Genome Consortium
liver cancer Japan (ICGC-LIRI-JP) (n = 240 tumor/202 normal), GSE14520 (n = 225/220), and GSE76427 (n = 115/52), and single-cell
RNA sequencing (scRNA-seq) datasets from GSE149614 (10 patients, ~93, 000 cells, four tissue sites) and GSE202642 (~74, 957 cells)
were retrieved from public repositories. EV transcriptome reference data were obtained from the EVmiRNA database and Vesiclepedia.
Integrative bioinformatic analyses included differential expression profiling of 23 m6A regulatory genes, EV/cellular RNA enrichment
ratio analysis, m6A site database cross-referencing, weighted gene co-expression network analysis (WGCNA), proteinprotein interaction (PPI) network construction and hub gene analysis, cell-type deconvolution, intercellular communication inference (CellChat), and
clinical outcome evaluation. Result: Fourteen of 23 m6A regulators were significantly dysregulated in HCC across all four cohorts, with
METTL3, WTAP, YTHDF1, YTHDF2, and IGF2BP1 showing the most consistent upregulation. Cross-referencing EV-enriched transcripts
with high-confidence m6A-modified sites identified a 63-transcript m6A-marked pro-metastatic EV cargo set, including VEGFA (EV/cellular enrichment = 3.8-fold), TGF-?1 (3.2-fold), HIF1A (2.9-fold), MALAT1 (4.1-fold), and SNAI1 (2.6-fold). YTHDF2 emerged as the
principal network hub (degree centrality k = 23, betweenness centrality rank 1) through physical interactions with TSG101 and PDCD6IP/
ALIX. The YTHDF2-correlated WGCNA turquoise module (1, 241 genes; r = +0.76 with YTHDF2; r = +0.71 with vascular invasion; both
p < 0.001) was enriched in TGF-?, VEGF, ECM-receptor interaction, and epithelialmesenchymal transition (EMT) pathways. Cell-type
deconvolution confirmed that YTHDF2-high HCC was associated with M2 macrophage enrichment, regulatory T cell (Treg) accumulation,
and CD8+ T cell depletion. Single-cell analysis showed progressive YTHDF2 induction from primary tumor to portal vein tumor thrombus
(PVTT) and lymph node (LN) metastatic compartments (mean expression: 2.34 ? 2.71 ? 2.89; both p < 0.001). An m6AEV axis signature score discriminated advanced-stage HCC (TNM IIIIV) with area under the receiver operating characteristic curve (AUROC) values
of 0.84, 0.79, and 0.77 in three independent cohorts, with hazard ratios of 2.17, 1.94, and 1.82 for overall survival (OS), respectively (all p
< 0.001). Conclusion: This integrative bioinformatic study establishes m6A modification as a molecular barcode governing selective RNA
cargo loading into HCC-derived EVs, with YTHDF2 as the central sorting hub acting through physical engagement with the endosomal
sorting complexes required for transport (ESCRT) machinery. YTHDF2 and the m6AEV cargo transcripts represent mechanistically
grounded targets for therapeutic intervention and non-invasive biomarker development in advanced HCC.
Keywords
N6-methyladenosine; Extracellular vesicles; RNA cargo sorting; Pre-metastatic niche; Hepatocellular carcinoma; Bioinformatics;
Epitranscriptomics
Full Text:
PDFReferences
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DOI: http://dx.doi.org/10.70711/pmr.v3i8.9533
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