The Mechanism Underlying The Regulation Of Mitochondrial Metabolism By Nuclear-encoded LncRNA NEAT1 That Aberantly Translocates To The Mitochondria Of Hepatocellular Carcinoma Cells

Background:Hepatocellular carcinoma(HCC)stands as one of the predominant malignant tumors globally.Despite the overall cancer mortality rate decreasing in the United States,the incidence of liver cancer in women continues to rise approximately 2%annually.Liver cancer is the second only to lung cancer in terms of cancer-related mortality.Over the past decade,substantial advancements have been achieved in the systemic treatment of advanced HCC.However,drug resistance remains a common issue among patients with HCC.While precision medicine offers the potential for enhanced treatment outcomes and reduced adverse effects,its application in HCC is still nascent due to the elusive molecular mechanisms underlying the disease.Consequently,there is a critical need for reliable biomarkers for early diagnosis and precise treatment.Mitochondria,the paramount energy source of cells,play a crucial role in both physiological and pathological cell processes.The malignant phenotype of tumor cells is also intimately linked to mitochondrial dysfunction.Long non-coding RNAs(lncRNAs)are known to regulate cellular functions and are associated with the genesis and progression of various tumors.LncRNAs have been identified as key regulators in a myriad of HCC biological behaviors,presenting potential as significant diagnostic,prognostic,and therapeutic tools for HCC.Recent discoveries have highlighted the role of mitochondria-associated lncRNAs in mediating interactions between the mitochondria and the nucleus,thereby maintaining intracellular stability.The nuclearencoded lncRNA NEAT1,a critical component of paraspeckles,participates in numerous biological functions and pathological processes,including the regulation of tumor cell behaviors.Serving as the molecular scaffold of paraspeckles,NEAT1 facilitates the communication between the mitochondria and the nucleus,thereby influencing cellular functions.To identify molecular factors that regulate mitochondrial function,we performed mitochondria RNA sequencing(mtRNA-Seq)between HCC cell line HepG2 and normal liver cells.Notably,we revealed a significant enrichment of lncRNA NEAT1 in the mitochondria of HCC cells.The role of mitochondrial lncRNA NEAT1 in tumor functionality remains to be elucidated.Objectives:This study was aimed to confirm the enrichment of nuclear-encoded lncRNA NEAT1 in the mitochondria of HCC cells.The impact of aberrantly translocated NEAT1 on the biological behavior and mitochondrial function of HCC cells was also examined.Finally,the molecular mechanism underlying the role of NEAT1 in the mitochondria of HCC cells was investigated.Methods:1.The enrichment of lncRNA NEAT1 in HCC cell mitochondria was confirmed via RT-qPCR.RNA fluorescence in situ hybridization(FISH)was utilized to ascertain the spatial localization of lncRNA NEAT1 within mitochondria.2.A novel LwaCas13a-mBarnase-MLS system was developed to specifically knockdown lncRNA NEAT1 in the mitochondria of HCC cells.3.Cell proliferation was measured using the CCK-8 assay and colony formation assay.Cell migration was assessed through the wound healing assay,and cell apoptosis via flow cytometry.The effect of mitochondrial lncRNA NEAT1 on mitochondrial function was evaluated by observing mitochondrial distribution and morphology with transmission electron microscopy,measuring ATP levels with an ATP kit and Seahorse technology,and detecting changes in mitochondrial membrane potential with JC-1 and MitoTracker Red CMXRo.4.The expression levels of respiratory chain complexes and ATPIF1 were measured by Western blotting,and ATP synthase activity was assessed using an ATP synthase activity detection kit.Co-immunoprecipitation(Co-IP)and immunofluorescence colocalization techniques were employed to detect the interaction of ATPIF1 with ATP.The RNA immunoprecipitation(RIP)method identified interactions between lncRNA NEAT1 and ATP synthase,as well as ATPIF1.The RNA pulldown method was used to determine the binding regions of NEAT1 with ATP synthase and ATPIF1.Online predictions using catRAPID identified potential binding sites of NEAT1 on ATP synthase and ATPIF1 proteinThe interaction sequence of NEAT1 with ATPIF1 protein was further validated through the RNase A protection assay.Results:1.We successfully developed the LwaCas13a-mBarnase-MLS targeting system that specifically knockdown mitochondrial RNAs.2.Targeted mitochondrial knockdown of lncRNA NEAT1 in HCC cells led to an increase in apoptosis without significant impact on cell proliferation and migration.In addition,we found that targeting mitochondrial NEAT1 induced relocalization of mitochondria from the cytoplasm to perinuclear regions.We also notived a reduction in ATP production,in parallel with an elevation in mitochondrial membrane potential.3.Despite the targeted knockdown of lncRNA NEAT1,the expression of the respiratory chain complex and ATPIF1 remained unchanged in HCC cells.However,ATP synthase activity was notably reduced.Further mechanistic investigations identified the interaction sites between lncRNA NEAT1 and ATPIF1,suggesting that aberrantly translocated NEAT1 functions through the ATP synthase-ATPIF1 axisi.Conclusions:1.The LwaCas13a-mBarnase-MLS mitochondrial RNA targeting system can successfully knock down the level of mitochondrial RNA without affecting its nuclear level.2.Translocation of nuclear encoded lncRNA NEAT1 to mitochondria may interact with ATPIF,potentially mitigating inhibition of ATPIF1 on ATP synthase,thereby promoting ATP production and cell apoptosis.