Nuclear Genome Encoding LncRNA MALAT1 Regulates Mitochondrial Metabolism-apoptosis-mitophagy In Hepatocellular Carcinoma By Abnormal Shuttling

Background: Mitochondria dysfunction has been widely accepted as the hallmark of cancer.Interestingly,several nuclear-encoded noncoding RNAs(nc RNAs)are newly discovered in mitochondria concerting with transcription factors and epigenetic regulators to modulate mitochondrial gene expression and mitochondrial function.MALAT1(metastasis-associated lung adenocarcinoma transcript 1),a highly conserved nucleus encoded long coding RNA(lnc RNA),dysregulated in multiple human malignancies is recently found to be related to mitochondria dysfunction.Methods: 1.Mitochondria isolation and RNA sequencing were conducted to find mitochondria-enriched lnc RNAs in hepatocellular carcinoma cell line Hep G2 and normal liver cell line HL7702.2.Through Reverse transcription-associated trap(RAT-seq),RNA immunoprecipitation(RIP),fluorescence in situ hybridization(FISH),MALAT1 interaction proteins and DNA were explored,and the distribution of MALAT1 in mitochondria was visualized.3.Silencing MALAT1 by sh RNA method,MALAT1 knockdown cell line metabolism,ROS amount,mitochondrial autophagy,apoptosis and mitochondrial morphological changes were detected by using Seahorse metabolic detector,ATP detection kit,mito SOX staining,mitochondrial SOD activity detection kit,lysotracker staining,flow cytometry(Annexin V/7-AAD staining),immunoblotting and transmission electron microscopy.4.The tumor biological behaviors including: cell proliferation assay,tumor clone formation capability,invasion,metastasis,cell cycle,cell apoptosis were measured by CCK-8 assay,colony formation assay,Transwell chamber assay,wound heal assay and flow cytometry.Results: By combining mitochondria-RNA transcriptome sequencing with FISH,we were surprised to discover that this nucleus DNA-encoded oncogenic lnc RNA was enriched in the mitochondria of Hep G2 cells.Knockdown of MALAT1 induced multiple abnormalities in mitochondrial functions,including low OXPHOS,low ATP production,reduced mitophagy,declined mt DNA copy number,and activation of the mitochondrial apoptosis pathway.Using RAT-seq approach,we found that MALAT1 lnc RNA utilized it 3'-fragment to interact with multiple loci of mitochondrial DNA(D-loop,COX2,ND3,and CYTB genes).Additionally,MALAT1 knockdown cell lines showed increased DNA methylation which correlated with decreased MT-DNA gene expression.The RIP and affinity RNA pulldown assays suggested that the RNA-binding protein Hu R may mediate the transportation of MALAT1 to the mitochondria.Additionally,mitochondria transmembrane protein mitochondrial carrier 2(MTCH2)was found to interacted MALAT1,suggesting that MALAT1 might get into the mitochondria through the MTCH2.Conclusions: In generally,this study greatly expands our knowledge of mitochondria function drived from the nucleus-encoded lnc RNA MALAT1.Our study established MALAT1 as a mitochondrial function regulator through interacting with MT-DNA,Hu R,MTCH2 protein,revealing a new regulatory mechanism of mitochondria.Many novel nucleus-encoded RNAs existing in mitochondria were identified for the first time,suggesting novel biological functions of lnc RNAs,laying the foundation for further clarifying the mitochondrial roles of lnc RNAs.