Hypoxia-driven metabolic reprogramming is a hallmark of hepatocellular carcinoma (HCC) and depends critically on mitochondrial signaling. We sought to identify RNA-based molecular factors that orchestrate the hypoxia-mitochondria crosstalk and regulate metabolic adaptation in HCC cells.An integrated mtRNA-seq and mitochondria-specific LwaCas13a-BN-MLS RNA targeting approach was employed to profile RNA molecules aberrantly enriched in HCC mitochondria. Mitophagy was assessed via mt-Keima assay, immunofluorescence, transmission electron microscopy, and Western blotting of key autophagic markers. RNA-protein interactions were examined using RNA immunoprecipitation (RIP), electrophoretic mobility shift assays (EMSA), and computational structural modeling.andtumorigenicity was evaluated using colony formation, transwell invasion, wound healing, and subcutaneous xenograft models in nude mice.Nuclear-encodedmRNA was aberrantly translocated to mitochondria, where it functions as a non-translating mRNA (ntmRNA) essential for mitophagy. Mitochondria-specifictargeting disrupted mitochondrial homeostasis by accumulating damaged mitochondria, lowering ATP, increasing ROS, reducing membrane potential, diminishing spare respiratory capacity, and impairing hypoxia-induced mitophagy. Mechanistically,ntmRNA promoted mitophagy through the HIF-1α/BNIP3/NIX axis by recruiting BNIP3 to mitochondria and coordinating its homodimerization via a 3'-UTR MRE. A synthetic MRE oligonucleotide rescued BNIP3 dimerization afterdepletion. Finally, we demonstrated thatloss suppressed malignant phenotypesand reduced xenograft tumor growth.This study reveals a noncanonical role formRNA as an epigenetic regulator of mitophagy in HCC, thus expanding the functional repertoire of mRNA molecules beyond protein coding. Targeting this noncanonicalntmRNA-BNIP3 homodimerization mechanism may suggest new therapeutic strategies for treating HCC. Background: Methods: Results: Conclusions: In vitro in vivo EGR1EGR1EGR1EGR1EGR1in vitro in vivo EGR1EGR1
