| Metabolic-associated fatty liver disease(MAFLD)is one of the most common chronic liver diseases,and its pathogenesis is related to lipid metabolism disorders,oxidative stress,and apoptosis,etc.Mitochondria are the main sites of cellular energy metabolism and participate in the regulation of physiological processes such as cell growth,oxidative phosphorylation and lipid metabolism.Therefore,their dysfunction can promote the abnormal accumulation of intracellular lipids,induce a series of events including oxidative stress,inflammation and cell apoptosis during the development of MAFLD,which make it the key to the transformation from fatty liver to steatohepatitis(NASH).Micro RNAs(miRNAs)are endogenous non-coding RNAs that are involved in the regulation of multiple signaling pathways in cells.Some miRNAs have been found to regulate mitochondrial function by targeting single or multiple mitochondrial proteins or mitochondrial-related factors,thereby delaying or aggravating MAFLD-related pathological process.Mi RNAs have certain advantages for the treatment of chronic diseases with complex pathogenesis due to their multitarget,endogenous and other characteristics.Moreover,with the gradual rise of small nucleic acid drugs,the development of miRNAs drugs with miRNAs regulating mitochondrial function for the treatment of MAFLD has important value.Screening of miRNAs regulating mitochondrial function in vitro: In the previous study,our laboratory used a cell model of palmitic acid(PA)-induced hepatocyte(L02)injury,and screened out 54 differentially-expressed miRNAs that have regulatory effects on reactive oxygen species(ROS)from GEO clinical datasets.Based on these,my research continued to screen and evaluate miRNAs that can regulate mitochondrial function.Since the JNK signaling pathway,links mitochondrial damage and hepatocyte apoptosis/inflammation as a downstream molecular event of ROS-induced mitochondrial dysfunction and ER stress,we firstly screened out 14 miRNAs that can regulate PA-induced p-JNK in L02 cells.Then,the effects of these miRNAs on mitochondrial function were evaluated from three aspects: mitochondrial oxidative phosphorylation capacity,mitochondrial membrane potential change,and mitochondrial DNA content.Finally,it was found that:(1)miR-337-3p and miR-675-3p can reduce the PA-induced p-JNK,and miR-337-3p can restore the damaged mitochondrial DNA content,while miR-675-3p can restore mitochondrial oxidation phosphorylation capacity and membrane polarization;(2) miR-612 and miR-15a-3p can increase the PA-stimulated p-JNK,aggravate the decrease of mitochondrial oxidative phosphorylation capacity and mitochondrial DNA content.Evaluation of the effects of miRNAs regulating mitochondrial function in vitro: We then evaluated the mitochondrial function-related effects of 4 miRNAs,miR-337-3p,miR-675-3p,miR-15a-3p,and miR-612.Firstly,the expression of these 4 miRNAs in the clinical datasets were analyzed,and the analysis results showed that these 4 miRNAs were differentially expressed in the pathogenesis of MAFLD,which is of significance for continued development: compared with the healthy group,the expression levels of miR-337-3p,miR-675-3p and miR-15a-3p in fatty liver group were downregulated;compared with the fatty liver group,the expression levels of miR-15a-3p and miR-612 in NASH group were upregulated significantly.After that,we used cleaved PARP and anti-apoptotic protein Bcl-2 as indicators of apoptosis,IL-6 and IL-1β as indicators of inflammation,and oil red O staining as indicators of lipid accumulation to evaluate the effects of these 4 miRNAs on mitochondrial functionrelated effects.Finally,it was found that:(1)miR-337-3p can reduce PA-induced hepatocyte apoptosis;(2)miR-15a-3p and miR-612 can aggravate PA-induced hepatocyte apoptosis,inflammation and lipid accumulation in L02 cells.Biological activity evaluation of miR-337-3p in vivo and in vitro: Given the protective effect of miR-337-3p obviously in vitro,we thus knocked out endogenous miR-337-3p in L02 cells by the CRISPR/Cas9 system to further detect the effect of miR-337-3p on mitochondrial function and MAFLD.The results showed that miR-337-3p-/-can aggravate the reduction of damaged mitochondrial oxidative phosphorylation capacity,apoptosis and lipid accumulation.Based on previous results of our laboratory in adeno-associated virus delivery of miR-337-3p on high-fat,highfructose-high cholesterol(HFHFr HC)-induced mice,we used PEG-P(DTC-co-TMC)-PEI1200 nanovesicle to delivery miR-337-3p and carried out its pharmacodynamic evaluation in vivo.After optimizing the concentration and time gradients of miR-337-3p delivered by the nanovesicle delivery system,the animal administration conditions were determined as miR-337-3p/PEG-P(DTC-co-TMC)-PEI1200 was subcutaneously injected at 1.5 mg/kg once every 7 days.Next,the effect of miR-337-3p on MAFLD was examined in mouse model pre-fed by HFHFr HC diet for 16 weeks.After 12 weeks,we found:(1)the content of miR-337-3p in the liver was increased to about 300 times compared with vehicle group,(2)the protein and m RNA levels of the reported target gene of miR-337-3p(STAT3)decreased slightly,(3)serum fructosamine was significantly reduced at 4 and 9 weeks,which was consistent with previous results of adeno-associated virus-based delivery of miR-337-3p.These results suggested that nanovesicles can deliver miR-337-3p to the liver to play some effects,but since it also showed some hepatotoxicity,it may be one of the reasons why the endpoint pharmacodynamics was not observed and needed further research to resolve.In conclusion,my thesis started from differentially-expressed miRNAs in clinic to screen and obtain that miR-337-3p and miR-675-3p have a protective effect on mitochondrial function in MAFLD,and miR-612 and miR-15a-3p can aggravate mitochondrial damage in MAFLD.Importantly,their biological activities correspond to their clinical relevance,which provides new candidate targets or active molecules for treating MAFLD in small nucleic acid drug development. |
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