The LEPIS-HuR-TMOD4 Axis Regulates Hepatic Cholesterol Homeostasis And Accelerates Atherosclerosis

BackgroundAtherosclerotic cardiovascular disease(ASCVD)are still an important cause of mortality in many countries,and atherosclerosis(AS)is a key pathological basis.Hypercholesterolemia is the most important causative factor for ASCVD.Even in patients who meet existing lipid-lowering criteria,there are still some patients with residual risk,so it is urgent to find new lipid-lowering targets to reduce AS-related mortality and morbidity.Long non-coding RNAs(lncRNAs)perform key functions in many cellular events and pathophysiological processes.In the process of AS,lncRNAs have regulatory effects on lipid metabolism,inflammatory cascade,cell proliferation of blood vessel,apoptosis,cell adhesion,cell migration and angiogenesis.More than 98%of the genes in the human genome do not have the ability to encode proteins,among which there are still a large number of non-coding RNAs(ncRNAs)to be discovered.Studies have found that the expression of PSRC1 in the liver regulates liver cholesterol metabolism-related genes,improve blood lipid levels,inhibit the formation of AS and enhance plaque stability.Transcriptome sequencing found that a PSRC1-mediated liver-specific lncRNA LEPIS was significantly down-regulated,so the researches of LEPIS may strengthen the theoretical foundation for finding more clinical lipid-lowering targets.Through bioinformatics analysis,we found that lncRNA LEPIS and Tropomodulin 4(TMOD4)are located on the same chromosome and have similar positions,and have overlapping fragments with TMOD4,so TMOD4 may be the target of LEPIS to exert biological effects.The literature studies have suggested that TMOD4 affects the intracellular cholesterol level.Therefore,LEPIS may affect cholesterol metabolism by targeting TMOD4,thereby affecting the progression of AS.Binding to RNA-binding proteins to regulate the transcription of targets is one of the common ways that lncRNAs play biological roles,and studies have suggested that human antigen R(HuR)can regulate the transcript of TMOD4.Therefore,we further investigated the association between LEPIS,HuR,and TMOD4.In summary,LEPIS may regulate the expression of TMOD4 through HuR,affect hepatic cholesterol metabolism,and then influence the process of AS.In order to clarify this hypothesis,this project intends to explore the effects of LEPIS on cholesterol metabolism,levels of lipids in plasma and AS from both in vivo and in vitro studies,and to explore the specific regulation of LEPIS on TMOD4.Methods and ResultsIn vivo studiesApoE-/-mice overexpressing LEPIS and TMOD4 were constructed by tail vein injection of adeno-associated virus,and the aortas of mice were extracted.Oil Red O staining,tissue immunofluorescence,cholesterol level determination,qPCR and Western Blot were used to clarify the effect of LEPIS and TMOD4 overexpression on the formation of atherosclerosis,liver cholesterol metabolism,and the expression of cholesterol metabolism-related genes in mice.Our study found that the overexpression of LEPIS and TMOD4 promoted the occurrence of AS,reduced plaque stability,increased the lipid level in plasma,reduced the level of hepatic cholesterol,down-regulate the expression of LDLR on the surface of hepatocytes,and promote the expression of PCSK9 in the liver.In vitro studies1.Lentivirus was transfected into HepG2 cells to construct a cell model overexpressing LEPIS and TMOD4,and its effects on the expression of LDLR,PCSK9 and intracellular cholesterol were detected,and the conclusion of the above in vivo study was verified again.In a cell model,the effects of overexpression of LEPIS and TMOD4 on LDLR,PCSK9 expression and intracellular cholesterol levels were examined;the regulation of TMOD4 by LEPIS was clarified.2.The RIP experiment explored the binding of LEPIS to TMOD4 or HuR,respectively.Cell immunofluorescence was used to detect the subcellular localization of HuR,and the protein expression levels of HuR in the nucleus and cytoplasm were detected to clarify the specific mechanism of LEPIS regulation of TMOD4.Furthermore,CLIP-qPCR experiments revealed the binding region of HuR to LEPIS.We found that LEPIS indirectly targets TMOD4 by binding' to HuR,which is inhibited by silencing of HuR.The subcellular localization of HuR suggests that overexpression of LEPIS promotes the translocation of HuR from the nucleus to the cytoplasm,and upregulates TMOD4 expression by enhancing the stability of TMOD4 mRNA,thereby exerting its targeted regulation of TMOD4.Furthermore,HuR binds to the 341-359 region of LEPIS.3.CHIP-qPCR verifies the binding site of TMOD4 and the downstream PCSK9 promoter,and transfects lentivirus or plasmid into HepG2 cells to construct a cell model overexpressing LEPIS and TMOD4 and a cell model overexpressing LEPIS+TMOD4 downregulation,and detecting HuR,TMOD4,PCSK9 transcription and translation levels,again to verify the effect of TMOD4 on PCSK9.The study found that TMOD4 has a binding site with the PCSK9 promoter,and overexpression of LEPIS promoted the expression of TMOD4,thereby increasing the level of PCSK9.During this process,the total transcription and translation levels of HuR in cells remained unchanged.ConclusionThis study confirmed that the overexpression of hepatic LEPIS stabilized the stability of TMOD4 mRNA by promoting the transfer of HuR from the nucleus to the cytoplasm,thereby enhancing the protein abundance of TMOD4;the up-regulated TMOD4 increased the expression level of hepatic PCSK9 by binding to the PCSK9 promoter,resulting in Impairment of hepatic cholesterol uptake,resulting in increased plasma LDL-C levels,promotes the occurrence and development of AS.