| BackgroundIn recent years,the incidence of nonalcoholic fatty liver disease(NAFLD)has increased and has gradually developed into the most common chronic liver disease.The specific mechanisms that currently lead to hepatic steatosis and the transition to severe liver disease are still not fully elucidated,thus limiting the discovery of drug targets and biomarkers that can be used to design effective therapeutic strategies.It can be seen that the in-depth study of the regulation mechanism of liver lipid deposition in NAFLD has important clinical significance for the early prevention and treatment of NAFLD.The serine/threonine protein kinase MST1(mammalian Ste20-like kinase 1,STK4)is a mammalian STE20-associated kinase that is highly conservation and widely present in various tissues.In the past,the research on MST1 mainly focused on its regulation in cell regulation,tumorigenesis and organ size control.In recent years,the research on MST protein kinase family has focused on its relationship with metabolic regulation,especially its relationship with liver metabolism regulation.Some literatures and previous studies of this group have confirmed that MST1 has an important regulatory effect on hepatic lipid metabolism and can be used as a new target for the treatment of NAFLD.Therefore,the regulation of targeting MST1 expression in the liver may become an effective disease intervention for NAFLD.There are two ways of regulation: hepatic and exogenous.With adipose tissue as a classic endocrine organ,it also plays an important role in maintaining the homeostasis of liver lipid metabolism.Therefore,we focus on the presence of other lipid-derived molecules in theadipose tissue to regulate the liver.MicroRNAs(miRNAs)are a class of small RNAs that negatively regulate gene expression by inhibiting the corresponding target mRNAs and have recently become key regulators of metabolism.Studies have been found that miRNAs can not only play biological functions in their own synthetic cells,but also be secreted by synthetic cells into the circulatory system and enter other tissues to regulate the biological functions of recipient cells.The form in which secreted miRNAs enters the circulatory system is mainly encapsulated in microvesicles,and microvesicles provide an important guarantee for the stability of miRNAs.Exosomes,as a class of natural lipid nanoparticles,they can stably transport their contents to adjacent or distant cells,thereby further modifying the gene expression,signal transduction and overall function of target cells.Therefore,exploring the specific mechanism of fat-derived miRNAs acting on the liver lipid metabolism target genes by means of exosome transfer,may become a new research approach for liver lipid deposition regulation.As explained above,the liver is an important organ for fat synthesis and storage.The balance between the synthesis and oxidative decomposition of fatty acids in the liver is an important mechanism of lipid metabolism,whether circulating miRNAs derived from adipose tissue can mediate liver lipid metabolism and affect the development of NAFLD by mediating the expression of MST1 gene in liver tissue.The answer to this question will provide new experimental evidence for the metabolic linkage of liver and adipose tissue,and provide new solutions and means for the treatment of NAFLD.Aim1.To explore the regulation of MST1 on hepatic lipid metabolism and its related mechanisms;2.Screening for miRNA molecules with high differential expression in mouse adipose tissue and targeting liver Mst1 gene in high fat background;3.To explore whether exosomes can carry specific lipid-derived miRNAs stably into hepatocytes and affect liver fat metabolism;4.Explore the possible signaling pathways and mechanisms by which specific adipose-derived miRNAs regulate liver fat metabolism.Materials and Methods1.Clinical liver samples from patients with clinical NAFLD and non-NAFLD,liver tissue samples from C57BL6 / J mice that were fed a high-fat diet or standard diet for 12 weeks.WB was used to detect the expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP1 c,pACC/ACC and FAS.qPCR was used to detect MST1 gene expression.2.Transfection of normal or PA-treated AML-12 cells with MST1 shRNA plasmid for 48 h.Oil red O staining was used to observe the accumulation of lipid droplets in liver tissue.Triglyceride content of hepatocyte was detected by enzyme colorimetric assay.The expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP 1c,pACC/ACC and FAS were detected by WB.The expression of MST1,SREBP 1c,ACC,FAS and CPT-1α was detected by qPCR.MSTLD model mice were injected with MST1 shRNA lentivirus via tail vein,anesthetized mice were taken one week after the injection.Lipid accumulation of liver tissue was observed by HE and oil red O staining.The content of TG,TC,HDL and LDL were detected.The expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP 1c,pACC/ACC and FAS were determined by WB.3.Transfection of normal or PA-treated AML-12 cells with pCDH-MST1 overexpressed plasmid for 48 h.Oil red O staining was used to observe the accumulation of lipid droplets in liver tissue.Triglyceride content of hepatocyte was detected by enzyme colorimetric assay.The expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP 1c,pACC/ACC and FAS were detected by WB.The expression of MST1,SREBP 1c,ACC,FAS and CPT-1α was detected by qPCR.MSTLD model mice were injected with MST1 shRNA lentivirus via tailvein.Lipid accumulation of liver tissue was observed by HE and oil red O staining.The content of TG,TC,HDL and LDL were detected.The expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP 1c,pACC/ACC and FAS were determined by WB.4.C57BL/6J male mice were sacrificed and fed with high-fat diet or standard feed for 12 weeks,and miRNAs targeting hepatic Mst1 gene were screened by RNA HiSeq/MiSeq sequencing in liver,adipose tissue and serum.Real-time PCR was used to detect the expression changes of miR-199 a in the above tissues.PA-treated or untreated AML-12 cells were transfected with miR-199 a mimics for 48 h.Oil red O staining was used to observe the accumulation of lipid droplets in liver tissue.Triglyceride content of hepatocyte was detected by enzyme colorimetric assay.5.miR-199a(XmiR-199a)or non-targeting miR-NT was loaded and transfected into exosome producing cells(HEK293)by using XMIRXpress lentiviral vector,followed by obtaining exosome miRNA by differential ultracentrifugation.WB was used to detect the exosome-associated protein markers CD9 and CD63 to confirm exosome characteristics.Exosome morphology was examined by electron microscopy.6.The RFP-labeled XPack plasmid was used to transfect exosome-producing cells(HEK293)for 48 h.Fluorescence microscopy detected HEK293 cells contained the reporter protein RFP,and thus can be used as a cell "factory" to secrete exosomes for downstream applications.Separation and purification of HEK293 cell culture medium The exosomes were added to AML-12 mouse hepatocyte culture medium,and the exosomes carrying the RFP-tagged protein were detected by fluorescence microscopy into the target cells.Exosome miR-199a(EXO-miR-199a)is injected into the nude mouse through the tail vein through GFP-tagged XMIRXpress lentiviral vector.The expression distribution position of miR-199 a in vivo was detected by fluorescence emission tomography(FLECT).The expression of miR-199 a in mouse liver was detected by RNA fluorescence in situ hybridization.7.Normal or PA-treated AML-12 cells were treated with 2 μg of EXO-miR-199 a orEXO-miR-NT for 48 h,and the expression of miR-199 a was detected by qPCR.Oil red O staining was used to observe the accumulation of lipid droplets in liver tissue.Triglyceride content of hepatocyte was detected by enzyme colorimetric assay.The MST1 3’ UTR region activity was detected by a dual luciferase reporter gene system.The expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP 1c,pACC/ACC and FAS were detected by WB.The expression of MST1,SREBP 1c,ACC,FAS,and CPT-1α were detected by qPCR.8.The NAFLD model was induced by high-fat injection of 30 μg EXO-miR-199 a or EXO-miR-NT in the tail vein for 3 weeks to detect changes in body weight,liver wet weight and serum triglyceride.H&E and oil red O staining were used to observe the accumulation of lipid droplets in liver tissue.Triglyceride content of hepatocyte was detected by enzyme colorimetric assay.The expression of MST1,pAMPK/AMPK,pSREBP 1c/SREBP 1c,pACC/ACC and FAS were detected by WB.The expression of MST1,SREBP-1c,ACC,FAS and CPT-1α was detected by qPCR.Results1.The expression of MST1 gene and protein in liver tissue with disordered lipid metabolism was significantly reducedThe protein level and mRNA level of MST1 in NAFLD patients were significantly lower than those in non-NAFLD patients;The protein and mRNA level of MST1 in the NAFLD animal model group were significantly lower than those in the NCD animal model group;The protein and mRNA level of MST1 in the NAFLD cell model group were significantly lower than those in the normal cell group.2.MST1 shRNA aggravates liver lipid synthesis.MST1 siRNA was transfected into PA-treated AML-12 cells for 48 h,oil red O staining,triglyceride determination,WB and qPCR results showed that SIRT1 siRNA significantly aggravated liver fat accumulation.In the high-fat-fed mouse model,MST1 shRNA lentiviruswas injected into the tail vein.MST1 shRNA significantly aggravated the fat accumulation effect by HE,oil red O staining and triglyceride content determination;MST1 shRNA significantly aggravated the fat accumulation effect;WB results showed that MST1 shRNA inhibited AMPK/SREBP 1c signaling pathway,induced ACC,increased FAS content,and aggravated fat metabolism disorder.3.MST1 overexpression reduces the synthesis of triglyceride in hepatocytes.Transient transfection of MST1 overexpressed plasmids in vitro and injection of MST1 overexpressed lentivirus in the tail vein in vivo,comprehensive HE staining,oil red O staining,and determination of triglyceride content,the results show that overexpression of MST1 significantly improved lipid metabolism in hepatocytes,thereby ameliorating high fat-induced NAFLD.4.miR-199a-5p is mainly expressed in adipose tissue and aggravates liver lipid production.Liver,adipose tissue and serum samples were taken from NAFLD model mice and negative control mice.RNA HiSeq / MiSeq sequencing and qPCR were used to confirm the high expression of miR-199a-5p,a characteristic targeting liver Mst1 gene,in adipose tissue,oil red O staining and triglyceride content determination showed that miR-199a-5p aggravated liver fat accumulation.5.Exosomes can wrap miR-199a-5p and directional transport to liver tissue.The WB results showed that the exosome marker proteins CD9 and CD63 were highly expressed in the EXO-miR-199 a group.The results of electron microscopy showed that the exosome of the bilayer membrane structure with a diameter of 30-100 nm was successfully separated by differential ultracentrifugation.Fluorescence microscopy showed that the exosomes carrying the RFP protein were successfully delivered to the target cell AML-12.Fluorescence emission tomography(FLECT)results and RNA fluorescence in situ hybridization in nude mice showed that EXO-miR-199 a was concentrated in liver tissue andstably expressed in liver tissue.6.EXO-miR-199 a aggravates liver lipid accumulation by targeting inhibition of liver MST1.2 μg of EXO-miR-199 a or EXO-miR-NT interfered with normal AML-12 cells for 48 h.qPCR results showed that the expression level of miR-199 a in EXO-miR-199 a group was significantly higher than that in EXO-miR-NT group.The results of oil red O staining and triglyceride content showed that the lipid droplet accumulation of hepatocytes in EXO-miR-199 a group increased significantly.2 μg of EXO-miR-199 a or EXO-miR-NT interfered with normal or PA-treated AML-12 cells for 48 h.The results of the dual luciferase reporter gene showed that the activity of fluorescein in the 3’UTR region of MST1 in the EXO-miR-199 a group was significantly lower than that in the EXO-miR-NT group,indicating that miR-199 a directly targets the inhibition of MST1 mRNA levels.WB showed that EXO-miR-199 a reduced the expression of MST1,pAMPK,pSREBP 1c and pACC,and increased the expression of FAS.qPCR results showed that EXO-miR-199 a significantly increased the expression of lipid-synthesis-related rate-limiting enzymes ACC and FAS and inhibited the expression of fatty acid oxidase CPT-1α.7.EXO-miR-199 a aggravates NAFLD through the MST1/SREBP-1c/AMPK pathway in vivo.30 μg EXO-miR-199 a or EXO-miR-NT was injected into the high-fat NAFLD model mice via the tail vein for 3 weeks,EXO-miR-199 a significantly increased the body weight and liver wet weight of mice.Liver morphology and staining showed that EXO-miR-199 a significantly aggravated lipid droplet formation in the liver.Serum and liver tissue triglyceride assays suggested that EXO-miR-199 a aggravated lipid-induced lipid abnormalities.The WB of liver tissue showed that EXO-miR-199 a inhibited the expression of MST1,pAMPK,pSREBP 1c,and pACC,and increased the expression of FAS.qPCR results showed that EXO-miR-199 a significantly increased the expression of lipid synthase and inhibited theexpression of fatty acid oxidase.Conclusion1.MST1 regulates liver lipid metabolism and alleviates liver lipid accumulation through AMPK/SREBP1 c pathway;2.miR-199a-5p is mainly expressed in adipose tissue and regulates hepatic lipid metabolism by exocrine function;3.Exosomes carrying miR-199a-5p can be transported into hepatocytes and promotes liver lipid synthesis;4.miR-199a-5p affects lipogenesis and lipolysis in the liver through the MST/AMPK/SREBP 1c pathway. |
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