The Mechanism Of Adipose-derived MiRNA Affecting Nonalcoholic Steatohepatitis By Targeting Liver MST1

BackgroundNonalcoholic fatty liver disease(NAFLD)is a common cause of chronic liver disease worldwide,with the incidence increasing year by year.If NAFLD progresses to non-alcoholic steatohepatitis(NASH)stage and is not intervened and treated,it will irreversibly develop into liver fibrosis,cirrhosis,and even hepatocellular carcinoma(HCC).With the progress of research,it has been found that cholesterol metabolism is closely related to the occurrence and severity of NASH.Therefore,in-depth study of the regulatory mechanism of liver cholesterol metabolism in NASH is of great significance for finding NASH drug treatment targets and early prevention and treatment of NASH.Mammalian Ste20-like kinase 1(MST1)is a core component of the Hippo signaling pathway and plays a key role in cell proliferation,immune response,cell apoptosis,and autophagy in various cellular biological processes.In recent years,the role of MST1 in regulating glucose and lipid metabolism has gradually received attention.Some literature and the previous research of our research group have found that MST1 has an important regulatory role in liver lipid metabolism,and liver lipid metabolism is closely related to cholesterol metabolism.Therefore,we believe that MST1 may also play a regulatory role in liver cholesterol metabolism.Adipose tissue is a classical endocrine organ.Under the background of obesity,adipose tissue and liver tissue can be regulated by micro RNAs(miRNAs)derived from adipose tissue by regulating gene expression in liver tissue.Adipose tissue is the main source of circulating miRNAs,and miRNAs are packaged in small vesicles and enter the circulation.Exosomes are lipid bilayer membrane vesicles with a diameter of 30-200 nanometers and have various activities,such as reshaping the extracellular matrix and transmitting signals and molecules to other cells.Therefore,exploring the mechanism of adipose-derived miRNAs targeting cholesterol metabolism genes in the liver through exosome transportation may become a new research area for regulating liver cholesterol metabolism.In summary,the accumulation of liver cholesterol is a key pathogenic factor leading to the development of NASH.With the background of the interaction between fat and liver tissue,exploring whether adipose-derived miRNAs can regulate liver cholesterol metabolism by regulating the expression of the MST1 gene in liver tissue and affect the occurrence and development of NASH may provide new research ideas for the drug treatment of NAFLD and theoretical basis for finding the best miRNA interference or miRNA target for NASH disease models.Aim1.To investigate the relevant mechanism of MST1 in the regulation of hepatic cholesterol synthesis and metabolism.2.To explore whether extracellular vesicles can transport specific fat-derived miRNAs to hepatocytes and affect hepatic cholesterol synthesis and metabolism.3.To investigate how specific fat-derived miRNAs regulate hepatic cholesterol metabolism and affect the progression of non-alcoholic steatohepatitis(NASH).Methods1.A cell model of NASH was established using free fatty acids(FFAs)as inducers at concentrations of 0.25 m M,0.5 m M,and 1 m M for 12,18,and 24 hours,respectively.After induction,Oil Red O staining,measurement of ALT and AST activity in the cell supernatant,detection of intracellular triglyceride content,and measurement of TNF-α level in the cell supernatant were performed to screen the cell model.2.C57BL6/J mice were fed a high-fat,high-cholesterol,and high-sugar diet for 18 weeks to establish a NASH mouse model.The model was validated by measuring the biochemical parameters in mouse serum,GTT,ITT,and liver pathology.Western blotting and q PCR were used to measure the protein and gene expression of MST1,AMPK,SREBP2,HMGCR,and HMGCS,as well as the gene expression of pro-inflammatory cytokines TNF-α,TGF-β,IL-6,IL-1β,and CCL2.The protein and gene levels related to the MST1/AMPK/SREBP2 pathway were validated by Western blotting,q PCR,and cell immunofluorescence staining experiments in both normal and NASH cell models.3.Hep G2 cells were treated with AMPK activator(AICAR),AMPK inhibitor(Compound C),transfection of MST1 sh RNA plasmid,and MST1 overexpression plasmid in normal or FFA-induced conditions.The cellular cholesterol and free cholesterol contents were measured by colorimetric assay,and the protein levels of p AMPK/AMPK,P-SREBP2/NSREBP2,HMGCR,and HMGCS1 were detected by Western blotting.The gene expression of pro-inflammatory cytokines TNF-α,TGF-β,IL-6,IL-1β,and CCL2 was measured by q PCR.The accumulation of free cholesterol in liver cells was analyzed by FILIPIN staining,and the localization and expression of SREBP2 in cells were analyzed by immunofluorescence staining.AICAR and Compound C were injected into NASH model mice intraperitoneally,and MST1 sh RNA lentivirus and MST1 overexpression lentivirus were injected into mice via the tail vein.The levels of ALT,AST,TC,and TG in mouse serum were measured,and the TC and FC contents in liver tissues were measured by colorimetric assay.The accumulation of free cholesterol in liver tissues was analyzed by FILIPIN staining.The protein levels of p AMPK/AMPK,P-SREBP2/N-SREBP2,HMGCR,and HMGCS1 were detected by Western blotting,and the gene expression of pro-inflammatory cytokines TNF-α,TGF-β,IL-6,IL-1β,and CCL2 was measured by q PCR.The degree of liver fat and inflammation was analyzed by HE staining.4.The human adipose-derived miR-518 a targeting liver MST1 was validated using a dual-luciferase reporter gene assay.The miR-518 a fragment was connected to the exosome packaging vector XMIRXpress and then transfected into HEK293 cells.Exosomes containing miR-518 a were extracted and verified using WB,TEM,and NTA.5.The connection vector Xpack-miR-518 a containing green fluorescent protein(GFP)was transfected into HEK293 cells.The exosomes in the culture medium of HEK293 cells were isolated and purified and then added to Hep G2 cells to observe whether Xpack-miR-518 a carrying the GFP tag could be delivered to the target cells via exosomes under fluorescence microscopy.6.Using exosomes loaded with miR-518a(EXO-miR-518a)to intervene normal or FFAcultured Hep G2 cells,cellular cholesterol levels were detected by colorimetric assay,and intracellular accumulation of free cholesterol was confirmed by FILIPIN staining.The protein and gene expression of MST1/AMPK/SREBP2 pathway were detected by WB and q PCR,and SREBP2 cellular localization was analyzed by immunofluorescence staining.Hsa-miR-518 a overexpressing lentivirus was intravenously injected into the normal diet group and NASH model group mice,and RNA fluorescence in situ hybridization was used to detect the expression of miR-518 a in mouse liver.The accumulation of free cholesterol in the liver tissue,liver inflammation and serum levels of ALT,AST,and TC in model mice were analyzed by detecting TC,FC content and FILIPIN staining in liver tissue.The protein and gene expression in MST1/AMPK/SREBP2 pathway and inflammatory factor genes were detected by WB and q PCR,and the degree of fatty and inflammatory liver lesions was analyzed by HE staining in liver tissue.Results1.FFA induced Hep G2 cells to construct NASH cell model After inducing Hep G2 cells with FFA at a concentration of 1m M for 24 hours,the activity of ALT and AST was the highest,TNF-α concentration was the highest,intracellular lipid deposition increased,and triglyceride content was the highest,achieving the best modeling effect.2.Western diet pattern constructs NASH animal model C57BL/6J mice were fed a Western diet(WD)consisting of high-fat,high-cholesterol,and high-sugar water simulation.After 18 weeks,the model mice showed impaired glucose tolerance,insulin resistance,abnormal liver function,elevated serum TC,TG,and GLU,significantly increased liver tissue TG content,liver enlargement,significant hepatic steatosis,inflammatory cell infiltration in liver tissue,and pathological changes in liver tissue consistent with NASH.3.MST1 protein and gene expression decreased,cholesterol accumulation increased in NASH liver model The results of WB in the NASH cell model group and the animal model group showed that compared with the normal control group,the expression of MST1 protein decreased,the expression of AMPK phosphorylation decreased,and the expression of NSREBP2 increased.q PCR results showed that the downstream regulated cholesterol synthesis genes HMGCR and HMGCS were upregulated,and the expression levels of related proinflammatory cytokine genes such as TNF-α increased.The content of TC and FC in liver tissue increased.4.AMPK inhibits the nuclear translocation and activation of SREBP2 to participate in regulating cholesterol synthesis metabolism.Upon treatment with Compound C,the WB results showed increased protein expression of N-SREBP2,HMGCR,and HMGCS1 in both WT and NASH cell models,while q PCR showed increased gene expression of SREBP2,HMGCR,and HMGCS,as well as elevated expression of pro-inflammatory factors such as TNF-α.Treatment with AICAR reduced the expression of N-SREBP2,HMGCR,and HMGCS1 proteins,as well as the expression of cholesterol synthesis genes and inflammatory factors.In NASH model mice treated with intraperitoneal injection of Compound C,serum ALT,AST,TC,and liver tissue TC and FC levels were higher than those in the control group,while in the AICAR group,these levels were reduced.WB and q PCR results were consistent with those in cell models,with increased expression of N-SREBP2 and upregulation of cholesterol synthesis genes in the Compound C group,accompanied by aggravated liver fat degeneration and inflammation.After treatment with AICAR,the expression of N-SREBP2 decreased,cholesterol synthesis pathway gene expression was suppressed,hepatic cholesterol deposition was reduced,and liver inflammation was alleviated.5.Knockdown of MST1 aggravates hepatic cholesterol accumulation and promotes NASH inflammation progression.WT and NASH cell models were transfected with MST1 sh RNA plasmids.The results showed that knockdown of MST1 inhibited AMPK activity,increased the expression of N-SREBP2,HMGCR,and HMGCS1 proteins,upregulated the expression of cholesterol synthesis-related genes and pro-inflammatory factors,and increased intracellular TC and FC content in both cell models.After knockdown of MST1 in WD model mice,N-SREBP2 expression increased,the cholesterol synthesis pathway was activated,serum TC increased,hepatic free cholesterol accumulation increased,and liver damage,fat degeneration,and inflammation worsened.6.Overexpression of MST1 can improve hepatic cholesterol deposition and reduce NASH inflammation levels.WT and NASH cell models were transfected with MST1 overexpression plasmids.Results from both cell models showed that after overexpression of MST1,the AMPK/SREBP2 pathway was activated,and the expression of N-SREBP2,HMGCR,and HMGCS decreased,as well as the expression of pro-inflammatory cytokine genes related to NASH.Cell immunofluorescence results showed that the localization expression of SREBP2 in the nucleus decreased while the expression outside the nucleus increased.The TC and FC content in the cells also decreased after overexpression of MST1.In the WD mouse group,after injection with MST1 overexpression lentivirus via tail vein,the serum ALT,AST,TC,and liver TC and FC content decreased in the overexpression group.WB and q PCR results showed that overexpression of MST1 could activate AMPK,inhibit the expression of genes related to cholesterol synthesis pathway,inhibit the expression of proinflammatory cytokines related to NASH,and alleviate hepatic inflammation in NASH model mice.7.Human adipose-derived miR-518 a can be packaged and transported by exosomes to liver tissue and target hepatic MST1.Dual-luciferase reporter gene assay results showed that hsa-miR-518 a significantly reduced the luciferase activity of MST1 3’UTR.After connecting the miR-518 a fragment with the exosome packaging vector XMIRXpress,exosomes were extracted,and WB detected exosome marker proteins CD9 and CD63,and electron microscopy observed exosome vesicles in circular or elliptical shapes,while NTA showed that most of the particles in the extract were between 30-200 nm in size.Under fluorescent microscopy,Xpack-miR-518 a labeled with GFP protein was observed to communicate between cells through exosomes and was successfully delivered to target cells Hep G2.8.miR-518 a exacerbates hepatic cholesterol accumulation and NASH inflammation by inhibiting the MST1/AMPK/SREBP2 pathway EXO-miR-518 a treatment was performed on both the WT and NASH cell models.The results of both cell models showed that after treatment with EXO-miR-518 a,the protein expression of MST1,p AMPK,and P-SREBP2 decreased,while the expression of N-SREBP2,HMGCR,and HMGCS1 increased.q PCR results showed that the expression of MST1 and AMPK genes decreased,while the expression of SREBP2,HMGCR,HMGCS,and related inflammatory cytokine genes increased.Additionally,the intracellular levels of TC and FC increased in the liver cells.After treating NCD and WD mice with a miR-518 a overexpression lentivirus via tail vein injection,the miR-518 a group exhibited increased serum levels of ALT,AST,and TC,while the WD group’s liver tissue showed a significant increase in TC and FC levels and FC deposition.WB and q PCR results showed that miR-518 a inhibited the expression of MST1 and p AMPK,promoted the translocation of SREBP2 into the nucleus,activated genes associated with the cholesterol synthesis pathway,increased the expression of pro-inflammatory cytokine genes in liver tissue,and exacerbated hepatic inflammation.Conclusion1.MST1 regulates hepatic cholesterol synthesis and metabolism through the AMPK/SREBP2 pathway,reducing cholesterol accumulation in the liver and alleviating NASH progression.2.Exosomes can transport fat-derived miR-518 a to the liver,targeting MST1 and affecting hepatic cholesterol synthesis and metabolism.3.miR-518 a exacerbates hepatic cholesterol accumulation and affects NASH progression by inhibiting the MST1/AMPK/SREBP2 pathway.