Nanoengineered polymeric RNA nanoflowers for dual-pathway modulation and effective therapy for non-small cell lung cancer

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality globally, with advanced stages posing a significant therapeutic challenge. In this study, we present a novel self-assembled RNA-based nanosystem that leverages rolling circle transcription (RCT) for concurrent inhibition of the long noncoding RNAs (lncRNAs) DB327252 and microRNA-21 (miR-21), two key regulators implicated in NSCLC pathogenesis. This system integrates RCT-derived siRNAs targeting DB327252 with circularized miR-21 decoys and self-assembles into cholesterol-decorated nanoparticles via hybridization of Chol-DNA anchor strands, thereby enhancing condensation, stability, and cellular uptake. The resulting nanoparticles exhibit superior bioavailability, stability, and effective intracellular delivery, facilitating the implementation of dual-pathway regulatory therapeutic strategies. Functional analyses demonstrated that the system significantly inhibited NSCLC cell proliferation, migration, and colony formation while promoting apoptosis. Mechanistically, simultaneous silencing of the lncRNA DB327252 and sequestration of miR-21 restored the expression of key tumor suppressors, including PTEN and PDCD4, at both the transcriptional and translational levels. In 3D NAC-Organ models composed of A549 cells and PBMCs, RNP-Chol treatment induced marked CD45 + immune cell infiltration, confirming its indirect immune-activating capacity. Furthermore, in vivo xenograft models validated the robust antitumor efficacy and high specificity of this nanosystem. Collectively, this RCT-enabled RNA platform provides a versatile and scalable approach for dual-pathway RNA therapeutics, with promising implications for precision medicine in NSCLC. • Self-assembled RCT-based RNA nanoplatform co-targets lncRNA DB327252 and miR-21. • Cholesterol-conjugated RNA/DNA hybrids enhance stability, condensation, and delivery. • Dual targeting restores PTEN/PDCD4, suppressing proliferation, migration, and inducing apoptosis. • NAC-Organ models recapitulate immune microenvironment with ATP depletion and immune activation.