Identification And Functional Study Of Circular RNA In Hepatic Steatosis

Objective:To uncover the expression profile of circular RNAs（circRNAs）,and the role of circRNA-0046367 in hepatocellular steatosis.Methods:HepG2 cells were randomized into groups of normal and model group,respectively.In contrast to the normal control group that treated by DMEM,model group was administrated by high-fat medium（OA: PA=2: 1,0.5mM）for 24 hours.Both oil red O staining and triglyceride（TG）assay were employed to evaluate the hepatocellular steatosis.Then the expression profile of circRNAs was investigated by microarray hybridization.Differentially expressed circRNAs were further subjected to validation by real-time quantitative polymerase chain reaction（real-time PCR）and functional analysis by hierarchical clustering,principal component analysis（PCA）,target prediction,gene ontology（GO）,and pathway annotation,respectively.Bioinformatic integration established the circRNA-miRNA-mRNA regulatory network.The interaction of circRNA-0046367-miR-34 a and miR-34a-PPARα related to circRNA-miRNA-mRNA regulatory network was bioinformatically investigated on the basis of complimentary binding,and then validated by dual luciferase reporter assay.Furthermore,the HepG2 cells were randomly divided into normal group（Normal）,high fat group（FFA）,vector control group（VC）,plasmid group（circRNA）,plasmid+mimics group（circRNA + mimics）and plasmid + mimics NC group（circRNA + mimics NC）,respectively.After the transfection of plasmid and/or mimics,the alternation of circRNA-0046367 and miR-34 a expression was analyzed by real-time PCR.The effect on miR-34a’s targets was evaluated by western blot.Both malondialdehyde（MDA）content and superoxide dismutase（SOD）activity were further detected to assess the lipid peroxidation and oxidative stress.Moreover,rhodamine-123 dying reflected the mitochondrial membrane potential（MMP）.Flow cytometry revealed the apoptosis rate.Results:In contrast to the control group,the model group exposed to high-fat stimulation exhibited significant increase in lipid droplets,and hepatic TG content [（24.4±6.4）μmol/g vs（183.7±27.5）μmol/g,P < 0.05].A total of 357 circRNAs was filtered to be differentially expressed during hepatic steatosis.The enrichment of transcription-related GOs,especially GO: 0006355,GO: 0000122,GO: 0045944,GO: 0045892,and GO: 004589,illustrated their specific function in transcriptional regulation.circRNA/miRNA/mRNA network also identified the signaling cascade of circRNA₀21412/miR-1972/LPIN1,which was characterized by decreased level of circRNA₀21412 and the miR-1972-based inhibition of LPIN1.The low expression of circRNA₀21412 down-regulated the transcription of long-chain acyl-CoA synthase（ACSL）,and finally led to hepatic steatosis.In addition,circRNA-0046367 was decreased in steatotic hepatocytes,while miR-34 a positively correlated with the severity of steatosis.The competitive binding of circRNA-0046367 and miR-34 a induced the up-regulated expression of peroxisome proliferator activated receptor alpha（PPARα）.Downstream genes related to lipid metabolism,including carnitine palmitoyltransferase 2（CPT2）and Acyl-CoA binding domain containing 3（ACBD3）,were transcriptionally activated.In result,HepG2 cells with normalized circRNA-0046367 demonstrated diminished TG deposition,lipid peroxidation,oxidative stress,and apoptosis.However,there was no protective effect on condition of the circRNA-0046367 saturation by miR-34 a mimics.Conclusion:The present study uncovers the circRNA expression profile specific to hepatocelluar steatosis,and constructs the circRNA/miRNA/mRNA regulatory network.The competitive complementation of circRNA-0046367 and miR-34 a rescues the PPARα expression,which further ameliorates the high-fat induced hepatocellular steatosis via transcriptional promotion of lipometabolic genes.Therefore,circRNA-0046367 is suggested to serve as the positive regulator,and potential therapeutic target of hepatic steatosis.