An lncRNA-aware single-cell framework with donor-level validation identifies reproducible MEG3 enrichment in human liver sinusoidal endothelial cells

Abstract

Long non-coding RNAs (lncRNAs) associated with metabolic liver disease are usually identified from bulk tissue, which cannot resolve the hepatic cell types that express them, and default single-cell pipelines discard most lncRNAs at feature selection. We present an lncRNA-aware single-cell analysis framework — retaining all detectable GENCODE v45 lncRNAs during highly variable gene selection — combined with donor-level validation that guards against pseudoreplication. The framework applies to already published data and needs no lncRNA-specific protocol. Applying it to the human Liver Cell Atlas (Gene Expression Omnibus accession GSE192742; 152,559 annotated cells, 16 donors), and using the source publication’s own cell-type annotation, we found MEG3 enriched in liver sinusoidal endothelial cells (LSECs) in abundance as well as in detection frequency: donor-level pseudobulk expression was 6.27 counts per 10,000 versus 1.05 in the next-ranked cell type, and the LSEC pseudobulk value exceeded the pooled non-LSEC value in all nine informative donors (paired Wilcoxon signed-rank: two-sided P = 0.0039, one-sided P = 0.0020). The LSEC-enriched detection pattern reproduced in an independent five-donor atlas (all five donors concordant). KCNQ1OT1 was not LSEC-specific. Benchmarking our clustering against the published annotation showed that two marker-scored lineages contained none of their nominal cell type; separately, the candidates returned by a within-lineage pseudotime screen were driven entirely by non-endothelial cells contaminating the LSEC lineage: correlations of |ρ| > 0.31 fell below 0.10 within correctly annotated endothelial cells and reversed sign in one quarter of alternative roots. We therefore report the pseudotime screen as a negative result, and provide the framework, the annotation audit and the two-cohort analysis as a reproducible resource.