Mitochondria-specific targeting of noncanonical <i>EGR1</i> ntmRNA-coordinated mitophagy receptor BNIP3 homodimerization disrupts mitochondrial metabolism and suppresses hepatocellular carcinoma growth <i>in vitro</i> and <i>in vivo</i>

<b>Background:</b> 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. <b>Methods:</b> 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. <i>In vitro</i> and <i>in vivo</i> tumorigenicity was evaluated using colony formation, transwell invasion, wound healing, and subcutaneous xenograft models in nude mice. <b>Results:</b> Nuclear-encoded <i>EGR1</i> mRNA was aberrantly translocated to mitochondria, where it functions as a non-translating mRNA (ntmRNA) essential for mitophagy. Mitochondria-specific <i>EGR1</i> targeting disrupted mitochondrial homeostasis by accumulating damaged mitochondria, lowering ATP, increasing ROS, reducing membrane potential, diminishing spare respiratory capacity, and impairing hypoxia-induced mitophagy. Mechanistically, <i>EGR1</i> 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 after <i>EGR1</i> depletion. Finally, we demonstrated that <i>EGR1</i> loss suppressed malignant phenotypes <i>in vitro</i> and reduced xenograft tumor growth <i>in vivo</i>. <b>Conclusions:</b> This study reveals a noncanonical role for <i>EGR1</i> mRNA as an epigenetic regulator of mitophagy in HCC, thus expanding the functional repertoire of mRNA molecules beyond protein coding. Targeting this noncanonical <i>EGR1</i> ntmRNA-BNIP3 homodimerization mechanism may suggest new therapeutic strategies for treating HCC.