Promoter hypermethylation-associated downregulation of LncRNA HAND2-AS1 in bladder cancer: functional and transcriptomic evidence

To investigate the expression pattern, epigenetic regulation, biological function, and transcriptomic associations of the long non-coding RNA (lncRNA) HAND2-AS1 in bladder cancer (BCa). HAND2-AS1 expression was examined using TCGA-BLCA data and validated by RT-qPCR in BCa cell lines and 28 paired tumor and adjacent bladder tissues. DNA methylation was evaluated by bisulfite sequencing PCR (BSP), and 5-aza-2′-deoxycytidine (5-AZA) was used as a pharmacological demethylation perturbation to assess methylation-sensitive HAND2-AS1 expression. Stable HAND2-AS1-overexpressing BCa cells were evaluated using CCK-8, Transwell, Annexin V/PI flow cytometry, and nude-mouse xenograft assays. RNA sequencing followed by GO, KEGG, Reactome, and protein–protein interaction analyses was used to characterize HAND2-AS1-associated transcriptomic changes. HAND2-AS1 expression was significantly reduced in BCa tissues and cell lines. BSP and the marked re-expression of endogenous HAND2-AS1 after 5-AZA treatment supported an association between promoter methylation and transcriptional repression. HAND2-AS1 overexpression inhibited BCa cell proliferation, migration, and invasion, increased apoptosis, and reduced xenograft tumor growth. RNA sequencing identified 149 differentially expressed genes (106 upregulated and 43 downregulated), with prominent enrichment of antiviral-response and interferon-related biological processes and pathways. In TCGA-BLCA, the univariable survival curve showed an expression-outcome association whose direction differed from the experimental tumor-suppressive phenotype, indicating that the clinical prognostic relationship is more complex than the cell-autonomous functional effect. HAND2-AS1 is downregulated in BCa and exhibits tumor-suppressive activity in experimental models. The combined methylation and 5-AZA findings support epigenetic repression as an important contributor to HAND2-AS1 downregulation. Transcriptomic profiling further links HAND2-AS1 overexpression to antiviral- and interferon-related gene programs. The clinical prognostic significance of HAND2-AS1 requires multivariable and independent-cohort validation.