Early Responding Gene EGR1 MRNA Noncoding Molecules Coordinate Mitochondria Functions Through The Hypoxia-mitophagy Pathway In Hepatocellular Carcinoma Cells

Background:Hepatocellular carcinoma(HCC)accounts for 75%-85% of primary liver cancers.It is one of the most common tumors worldwide and is ranked as the second most deadly malignant tumor in men.Unfortunately,most HCC patients are already in the advanced stage when they are diagnosed and thus cannot be treated with surgery.Additionally,the therapeutic effects of chemotherapy and radiotherapy are poor.In the past few decades,the 5-year survival rate of HCC patients has not improved significantly.Uncontrolled cell proliferation and metastasis are the main obstacles to clinical treatment and one of the causes of high mortality.Therefore,the discovery of molecular pathways for tumor progression is important for the identification of biomarkers for early detection and the development of new therapeutic strategies against HCC.As an important cell stress sensor,mitochondria are important performers of a series of activities such as the production of adenosine triphosphate(ATP)and ROS by the oxidative phosphorylation system.Mitochondria are crucial organelles for energy production and cellular homeostasis in mammalian cells.The maintenance of a healthy mitochondrial network is critical in the development as well as in the response to physiological adaptations and stress conditions throughout life.The role of mitochondrial in carcinogenesis and cancer progression is an area of active research,with many unresolved questions.Various aspects of altered mitochondrial function have been implicated in tumorigenesis and tumor progression,including mitochondrial dysfunction,a metabolic switch to aerobic glycolysis,and dysregulation of mitophagy.Mitophagy is a highly specific quality control process which eliminates dysfunctional mitochondria and promotes mitochondrial turnover and is involved in the adaptation to nutrient stress by controlling mitochondrial mass.Increasing evidence from various studies supports the notion that dysregulation of mitophagy is an etiological factor in tumorigenesis.Through mitochondria RNA sequencing(mt RNA-seq)to find molecules involved in the abnormal regulation of HCC,we were surprised to find that the early growth response protein 1(EGR1)encoded by the nucleus is enriched in the mitochondria of HCC cells.EGR1,an m RNA encoded by the nuclear genome,is a tumor suppressor gene that plays a key role in many biological processes in the nucleus,especially in response to ischemia and hypoxia.However,how EGR1 shuttles into mitochondria,how it plays a regulatory role in mitochondria,and whether it ultimately affects the outcome of HCC have not yet been addressed.Objectives:1.To reveal the mechanisms underlying the aberrant shuttering of EGR1 m RNAs from the nucleus into the mitochondria,as well as its enrichment and its function in the mitochondria of HCC cells.2.To clarify how the aberrantly shuttered EGR1 m RNAs regulate mitochondrial functions in HCC.3.To clarify the correlation between the mitochondria-enriched EGR1 m RNA and the occurrence and development of HCC,and whether it can be used as a new molecular biomarker for prognostic judgment and curative effect evaluation of HCC,so as to provide new research strategies for precise treatment of HCC.Methods:1.RNA-seq was performed to find mitochondrial RNA differentially expressed in HCC and normal liver cells.The distribution and content of EGR1 m RNA in mitochondria were further confirmed by Fluorescence in situ hybridization(FISH)and RT-q PCR.2.Immunofluorescence(IF)and Western blotting were used to determine if the mitochondria-enriched EGR1 m RNA can be translated into EGR1 proteins using the mitochondrial translation mechinery in HCC cells..3.The influence on mitochondrial function(such as ATP production,ROS,mitochondrial membrane potential,etc.)was detected between sh EGR1 group and sh Ctrl group.4.The effect of the mitochondria-enriched EGR1 mRNA on hypoxia-induced mitophagy was detected by transmission electron microscopy(TEM)and IF.5.Western blotting was used to examine the mitophagy pathway proteins as regulated by the mitochondria-enriched EGR1 m RNA6.The regulation of the mitochondria-enriched EGR1 m RNA on the formation of autophagosomes and mitochondrial dynamics(mitochondria fission and fusion)was determined by western blot and other methods.7.After EGR1 was knocked down by sh RNA,CCK-8 assay,Transwell chamber assay,scratch assay,plate clone formation assay and Annexinv/Sytox staining were performed to evaluate the effects of EGR1 on tumor cell proliferation,tumor clone formation ability,apoptosis,migration and invasion,etc.8.Through the mitochondria-specific Lwa Cas13 a RNA editing technology,we constructed the mitochondrial-localized Lwa Cas13 a RNA plasmid vector to specifically knockdown EGR1 in the mitochondria of HCC cell lines.Using this system,we further clarify the influence of EGR1 on mitochondrial function and hypoxia-induced mitophagy.Results:1.The nuclear-encoded m RNA EGR1 was aberrantly enriched in mitochondria of hepatoma cells.2.EGR1 m RNA functions in the mitochondria in the form of non-coding RNA.By isolating the mitochondria and performing western blotting,we found that EGR1 m RNA did not translate protein in mitochondria.3.Loss of EGR1 led to the accumulation of damaged mitochondria,which are manifested by decreased ATP production,decreased membrane potential,and increased reactive oxygen production.4.TEM showed that the number of autophagy lysosomes induced by hypoxia was reduced in the EGR1 knockdown group compared to the control group.Mitochondrial autophagy was significantly reduced in the EGR1 knockdown group by IF.These results indicated that the mitochondria-enriched EGR1 m RNA is essential for mitophagy in HCC.5.The mitochondria-enriched EGR1 noncoding mRNA molecules affected the recruitment of mitophagy receptors BNIP3 and NIX to mitochondria and promoted mitophagy through the HIF1?/BNIP3/BNIP3 L pathway.6.Mitochondrial EGR1 m RNA is essential for autophagosome formation during mitophagy and it is required for hypoxia-induced mitochondrial fission and negatively regulates mitochondrial fusion.7.The mitochondria-enriched EGR1 mRNA plays an important role in promoting tumor occurrence and development in hepatocellular carcinoma.8.Through Lwa Cas13 a RNA editing technology,we successfully constructed the mitochondrial-localized Lwa Cas13a-BN-MLS plasmid vector to specifically knockdown EGR1 in the mitochondria of HCC cell lines without affecting EGR1 in the nucleus.The experimental results were basically consistent with the results of EGR1 knockout at the whole cell level using sh RNAs,suggesting that The mitochondria-enriched EGR1 m RNA is necessary for mitophagy.Conclusions:1.In this study,we identified the mitochondrial enrichment of the nuclear genome-encoded EGR1 m RNA in Hep G2 cells compared with normal hepatocytes HL7702.Our study thus suggests a novel non-canonical genetic pathway that the EGR1 m RNA may have a noncoding function in mitochondria,in addition to its coding role through the ribosome.Hence,EGR1 plays an essential role in maintaining a healthy mitochondrial network through the tight coordination of the genetic messages between the nucleus-ribosome-mitochondria.2.Knockdown of EGR1 in mitochondria,whether using the LwaCas13aBN-MLS mitochondria-specific RNA targeting or sh RNA,significantly inhibits the mitophagy in HCC cells.As expected,depletion of EGR1 leads to the accumulation of damaged mitochondria,in parallel with mitochondrial dysfunctions,including decreased ATP production,increased ROS production,and decreased mitochondrial membrane potential.This study provides the first evidence that in addition to its well-known canonical peptide coding function,the nuclear genome-encoded EGR1 m RNA may play a non-canonical role in regulating mitophagy through the noncoding m RNA pathway.3.After EGR1 knockdown,we found that the migration and invasion ability of HCC cells was markedly weakened.At the same time,cell colony formation was significantly reduced,and cell proliferation was slowed down.Thus,it seems that The mitochondria-enriched EGR1 m RNA may play a role in promoting tumor genesis and development in hepatoma cells.