Extracellular Vesicle-Packaged <i>MIR4435-2HG</i> Facilitates Cigarette Smoke-Induced Bladder Cancer Progression through Enolase 1-Dependent Glycolytic Reprogramming

Epidemiological studies have reported that cigarette smoking promotes bladder cancer progression, but the corresponding biological mechanisms must be elucidated. Cigarette smoking-related extracellular vesicle (EV)-packaged long noncoding RNAs (lncRNAs) derived from bladder tumors were identified via RNA sequencing, tissue microarrays, and single-cell RNA sequencing. The clinical value of candidate EV-packaged lncRNAs was evaluated in the urine and plasma of bladder cancer patients with smoking history. The underlying mechanism of EV-packaged lncRNAs was explored using CRISPR/Cas9, Seahorse, and N4-acetylcytidine (ac4C) acetylation experiments <i>in vivo</i> and <i>in vitro</i>. The EV-packaged lncRNA <i>MIR4435-2HG</i>, which was originally secreted by M2 macrophages in response to exposure to the cigarette smoking-related carcinogen 4-aminobiphenyl (4-ABP), exhibited an abundant expression pattern. Mechanistically, 4-ABP promoted M2 macrophage polarization and increased fused in sarcoma (FUS) expression by inducing signal transducer and activator of transcription 6 (STAT6) phosphorylation, contributing to the direct packaging of <i>MIR4435-2HG</i> into M2 macrophage-derived EVs and subsequent delivery to recipient tumor cells. The nuclear EV-packaged <i>MIR4435-2HG</i> subsequently bound <i>N</i>-acetyltransferase 10 (NAT10) and increased the stability of the glycolysis regulator Enolase 1 (ENO1) through the ac4C modification; cytoplasmic EV-packaged <i>MIR4435-2HG</i> sponged miR-143-3p, increased ENO1 expression, and ultimately activated PI3K-Akt signaling for glycolytic reprogramming to promote tumor development. In addition, recipient tumor cells internalized EV-packaged <i>MIR4435-2HG</i> and simultaneously secreted chemokines to recruit monocytes, establishing a potential feed-forward loop between M2 macrophages and tumor cells. This study identified EV-packaged <i>MIR4435-2HG</i> as a crucial bladder cancer marker that mediates intercellular communication during cigarette smoke exposure, suggesting a promising approach for bladder cancer prevention and treatment.