Identification And Mechanism Study Of A Novel Long Non-coding RNA Lnc-HC In Cholesterol Metabolism

Objective:The disorders of lipid and cholesterol metabolism are essential clinical manifestations of metabolic syndrome.The long non-coding RNAs(lnc RNAs)have been reported to take part in the regulation of cholesterol metabolism.Hence,our study aimed to identify the critical lnc RNA which is important in cholesterol metabolism and clarify the regulation both in vitro and in vivo.Then,we attempted to make clear the molecular mechanism of lnc RNA in regulating gene expression and biological process.Finally,we would find out the upstream pathway of lnc RNA in cholesterol metabolism.This study was performed to provide the basis for the molecular pathogenesis research of cholesterol metabolism and metabolic syndrome.Methods:1.Suppression subtractive hybridization technology(SSH)was used to construct the subtractive c DNA libraries of differentially expressed genes in high-fiet diet induced metabolic syndrome(HFD-Met S)E3 rat livers.Twenty positive clones were randomly collected from forward c DNA library and were sent to sequencing,NCBI Blast and finally RT-q PCR to confirm the expression level.2.By using in silico cloning and SMART RACE technology,the full-length of the novel EST was obtained and named lnc-HC.To figure out whether lnc-HC is non-coding RNA or not,informatics analysis and experimental methods were used.3.Northern blotting and RT-q PCR were used to definite the relationship between lnc-HC and its overlapping protein-coding gene Slc25a15.In addition,the expression profile of lnc-HC in rat liver and different cell types were detected by using PCR and Northern blotting.4.To explore the cellular function of lnc-HC,the lnc-HC overexpression and knockdown experiments were conducted in CBRH-7919 cell lines.Then the critical enzymes related to cholesterol metabolism were detected both on m RNA and protein levels.Further,high-cholesterol diet(HCD)induced dyslipidemia rat model was induced to study the lnc-HC function in vivo.5.By using RNA pull-down,RIP and MS technology,the molecular mechanism of lnc-HC in regulation cholesterol metabolism was clarified.6.High cholesterol cell model was induced to detect lnc-HC expression level.Then,the core promoter region of lnc-HC was found out by using recombinant plasmids construction and dual luciferase reporter assay.For further identification of the specific reason for lnc-HC up-regulation in high level of cholesterl,informatics analysis,expression recombinant plasmids construction and dual luciferase reporter assay were used.7.Finally,the treatment of LXR? agonist T0901317 in CBRH-7919 cell lines was performed to make sure the mediation between high level cholesterol and C/EBP? expression and then lnc-HC.Results:1.The subtractive c DNA libraries of differentially expressed genes in HFD-Met S have been successfully constructed.After the sequencing and RT-q PCR confirmation,9 genes and 1 unannotated EST were identified as up-regulated in forward library.2.The full-length of lnc-HC is 1,410 nt.Informatics analysis results suggest that lnc-HC shows a lower conservation and a higher synteny among various species genomes and it lacks the Kozak sequence.The predicted ORF of lnc-HC does not match any existing peptides.In addition,we cloned the predicted ORF and transfected the expression plasmids into CBRH-7919 cell lines and no fusion protein was detectable.Thus,these results suggest that lnc-HC is a lnc RNA.3.lnc-HC is an independently transcribed lnc RNA with no association with the Slc25a15 gene.lnc-HC expresses in metabolic organs including liver,pancrease,fat and muscle.lnc-HC localizes in the nucleus of hepatocyte.4.The lnc-HC overexpression resulted in the decrease of Cyp7a1 and Abca1.The knockdown of lnc-HC significantly increased the expression of Cyp7a1 and Abca1.The knockdown of lnc-HC strongly recovered the glucose tolerance disorder and serum lipid disorders in vivo.5.hn RNPA2B1 is the protein partner of lnc-HC.hn RNPA2B1 could directly bind to Cyp7a1 and Abca1 m RNAs and reduce the two m RNAs expression.6.The proximal promoter region of lnc-HC is from-1,000 to-200 based from TSS.The up-regulation of lnc-HC by cholesterol treatment is mediated by the transcription factor C/EBP? binding to the lnc-HC proximal promoter region.7.The intracellular cholesterol accumulation up-regualtes the C/EBP? expression through activating LXR?,and the activated C/EBP? then enhance the transcription level of lnc-HC.Conclusion:1.lnc-HC is a novel lnc RNA,which is up-regulated in HFD-Met S rat livers.2.lnc-HC reverse-regulates the cholesterol catabolism.The lnc-HC overexpression within hepatocytes recruites hn RNPA2B1 into nucleus and directly interacts with hn RNPA2B1 through the 3' stem-loop,and the RNA-protein complex combines with target m RNA Cyp7a1 or Abca1 to form a RNA-protein-m RNA complex and induces the degradation of target m RNAs.3.The intracellular cholesterol accumulation increases the C/EBP? expression through activating LXR?,then the transcription of lnc-HC is activated by C/EBP? binding to the lnc-HC promoter region.