Lian-Jia-San-Jie formula suppresses non-small cell lung cancer via FTO-dependent m6A demethylation regulation of lncRNA CASC9 and its downstream CD3EAP

Background Dysregulation of the epitranscriptome is implicated in the aberrant expression of oncogenes during oncogenesis. In this study, we sought to elucidate the therapeutic mechanisms of Lian-Jia-San-Jie-Fang (LJSJF), a traditional Chinese medicinal formula composed of 14 herbs with Scutellaria barbata D. Don (whole herb) as the principal component, together with Sarcandra glabra (Thunb.) Nakai (whole herb), Ranunculus ternatus Thunb. (whole herb), Adenophora tetraphylla (Thunb.) Fisch. (root), Glehnia littoralis F.Schmidt ex Miq. (root), Pseudostellaria heterophylla (Miq.) Pax (tuberous root), Ophiopogon japonicus (Linn. f.) Ker Gawl. (root), Asparagus cochinchinensis (Lour.) Merr. (tuberous root), Rubia cordifolia L. (root and rhizome), Pinellia pedatisecta Schott (tuber), Hedyotis diffusa Willd. (whole herb), Bombyx batryticatus (dried larva), and Inula japonica Thunb. (flower) at a fixed clinical mass ratio, prepared by water extraction, concentration, and lyophilization. Methods We employed a zebrafish xenograft model of lung cancer to quantify cell proliferation and migration. Utilizing bioinformatic tools, we dissected the altered expression patterns of long non-coding RNAs (lncRNAs) within tumor cells following treatment with Lian-Jia-San-Jie-Fang (LJSJF). We further examined the expression levels and prognostic significance of CASC9/CD3EAP in tumor progression using a curated public database of tissue samples. To mechanistically probe the role of m 6 A modification on CASC9/CD3EAP axis upon LJSJF treatment, we performed m 6 A dot blot, MeRIP-qPCR and mRNA stability assays combined with Western blot analysis. Results LJSJF exerted dose-dependent anti-proliferative and anti-migratory effects in zebrafish NSCLC xenografts. In-vivo quantitative PCR with human-specific primer pairs was performed on whole zebrafish larvae. Although no off-target amplification from zebrafish genome was detected, these measurements reflect total human-derived transcript abundance in whole-larva lysates rather than per-cell gene expression within tumor cells. The results showed that total transcript levels of human CASC9 and CD3EAP were reduced in a dose-dependent manner in LJSJF-treated xenograft-bearing larvae. RNA-seq screening revealed widespread dysregulation of lncRNAs upon LJSJF intervention, among which oncogenic CASC9 was overexpressed in human lung tumor tissues and linked to unfavorable overall survival. We verified a positive correlation between CASC9 and CD3EAP expression, and high CD3EAP also predicted shorter patient survival. Mechanistically, FTO-mediated m 6 A demethylation epigenetically maintains high CASC9 levels to drive malignant progression, and this regulatory axis is blocked by LJSJF. Conclusion This study reveals that LJSJF restrains NSCLC malignant phenotypes through epigenetic repression of the FTO-CASC9-CD3EAP axis, providing a potential therapeutic target for NSCLC intervention.