Role And Mechanism Of Hsa-SETD2＿0009 In Lipid Metabolism Of Metabolic Dysfunction-Associated Fatty Liver Disease

Objective: In order to elucidate the role and regulatory mechanism of circRNAs in the development and progression of metabolic-associated fatty liver disease(MAFLD),based on the transcriptomic analysis of circRNAs from MAFLD patients,we screened out circRNAs differentially expressed in MAFLD and normal liver tissues and verified their expression in the liver tissues of MAFLD patients.Subsequently,we established MAFLD cell and animal model to explore the specific function and regulation mechanism of circRNAs in vivo and in vitro through a series of molecular biology experiments.Methods: High-throughput sequencing and transcriptomic analysis of liver tissues from MAFLD patients and healthy controls(HC)were performed to obtain the differential circRNAs expression profiles between the two groups.Subsequently,multiple gene function enrichment analysis,circRNAs function prediction and database control were used to screen the circRNAs most likely to play a role in MAFLD,q RT-PCR was used to detect the differential expression of circRNAs in liver tissues of MAFLD patients and HC Group.The ring-forming properties of the target circRNAs were verified by ring-forming experiments.The expression of circRNAs in MAFLD cell model induced by free fatty acids(FFA)was confirmed by q RT-PCR and agarose gel electrophoresis.To construct a Hep G2 cell line with stable and specific knockdown or overexpression of circRNAs by RNA interference,and then establish a MAFLD cell model,the changes of lipid content in the MAFLD cell model were observed by oil red O staining,Lipi-red fluorescence staining,and total triglyceride concentration determination after the intervention of target circRNAs,q RT-PCR and WB were used to detect the expression of key genes and proteins involved in lipogenesis and fatty acid β oxidation.Through RNA pull-down test and protein mass spectrometry analysis,the specific binding proteins of circRNAs were screened out,and the direct binding of circRNAs to protein was verified by RNA immunoprecipitation(RIP).The relationship between the expression amount of target circRNAs and target proteins in MAFLD cell model was detected by q RT-PCR and WB,and target proteins were activated or blocked in cell lines that specifically knockdown or overexpress target circRNAs,after the establishment of MAFLD cell model,we observed the changes of lipid content and metabolism in the third step to verify the interaction between them.A high fat diet(HFD)fed Apo E knockout C57BL/6J male mouse model of MAFLD was constructed,the expression of circRNAs and target protein in the animal model was verified by q RT-PCR,WB and immunofluorescence staining.Mouse models of specific knockdown or overexpression of target circRNAs in the liver were established by tail vein injection of the viral vector,the changes of hepatic steatosis in MAFLD mice after intervention of target circRNAs were observed by HE staining and oil red O staining of liver tissue sections.The changes of hepatic function,blood glucose and blood lipid were detected by serum biochemical indexes.q RT-PCR and WB were used to detect the expression of key genes and proteins involved in lipogenesis and fatty acid βoxidation,and to observe the changes of lipid metabolism in liver tissue of mice.Results: Sequencing and transcriptomic analysis of liver tissues from MAFLD patients and matched HC groups revealed that 59 circRNAs were differentially expressed,compared with HC Group,MAFLD group had 35 up-regulated genes and24 down-regulated genes.Four circRNAs which were significantly up-regulated in MAFLD were successfully identified in liver tissues of patients with MAFLD and HC Group.circSETD2 was identified by bioinformatics analysis,comparison of circAtlas database and analysis of the effect of its source genes.circSETD2 is a circRNAs with a closed loop and localized in the cytoplasm,which is stably expressed in the liver tissues of MAFLD patients and is significantly up-regulated compared with the HC Group.The expression level of circSETD2 in MAFLD cell model was significantly higher than that in control group,compared with the control group(NC),the MAFLD cell model with stable knockdown of CIRCSETD2 showed a significant decrease in intracellular lipid content,lipogenesis and fatty acid β oxidation.A total of 21 circSETD2-binding proteins were identified by RNA pull-down test and mass spectrometry.A total of 21 circSETD2-binding proteins were identified by RNA pull-down test and mass spectrometry analysis.The target protein was identified as CPS1 by protein reliability score and reliability rating.RIP experiment proved that circSETD2 and CPS1 could be directly combined in MAFLD cell model.Inhibition of CPS1 cell activity in a MAFLD model constructed from a stably knockdown circSETD2 cell line could significantly attenuate circSETD2 knockdown-generated cellular steatosis-mitigating effects.The expression level of mmu-circSETD2,a mouse homologous gene of circSETD2,was significantly higher in the liver tissue of MAFLD mice than that of the control group,and the expression level of CPS1 gene and protein was significantly lower than that of the control group.The expression of mmu-circSETD2 was successfully knocked down specifically in the liver of a MAFLD mouse model by tail vein injection of an sh-RNA viral vector that specifically knockdown mmu-circSETD2.When mmu-circSETD2 was specifically knocked down in the livers of MAFLD mice,the Steatohepatitis of the mice was significantly reduced,the lipogenesis of the liver tissue was significantly reduced,the liver function damage was significantly alleviated,and the level of blood lipids was significantly reduced,at the same time,the oxidation of fatty acid β increased significantly.After mmu-circSETD2 was specifically knocked down in the liver of MAFLD mice,the expression levels of CPS1 gene and protein were significantly up-regulated as compared with NC Group.Conclusion(s): circSETD2 is a circRNAs stably expressed in the liver of patients with MAFLD and significantly upregulated in comparison with HC Group,possessing a closed loop structure and localized in the cytoplasm.Functional tests in vitro showed that abnormal upregulation of circSETD2 promoted the disorder of lipid metabolism in MAFLD.Further mechanistic studies revealed that circSETD2 was involved in regulating the disorder of lipid metabolism and disease progression of MAFLD by reducing the expression of CPS1 and directly binding to CPS1 protein to reduce its cellular activity.Functional tests in vivo of circSETD2 in MAFLD mice showed that circSETD2 could regulate the development of MAFLD by reducing the expression of CPS1,aggravating the steatohepatitis,liver function damage and hyperlipemia in mice.circSETD2 can aggravate the disorder of lipid metabolism and disease progression of MAFLD,it is a potential prognostic marker and therapeutic target.