Role And Mechanisms Of Mitochondrial-cytoplasmic Redistribution Of MiRNAs In The Pathogenesis Of Non-Alcoholic Fatty Liver Disease

Background:Non-alcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by excessive intrahepatocellular fat deposition that occurs in addition to definite liver damage factors such as alcohol,drugs,and viruses.NAFLD not only significantly increases morbidity and mortality from liver-related diseases,but is also closely associated with the development of a variety of extrahepatic diseases.MicroRNAs(miRNAs)are mainly distributed in the cytoplasm of cells and several subcellular organelles,and are characterized by a distinct regional distribution,which plays an important role in maintaining normal cellular functions.However,the mechanisms underlying the regionalized distribution of intracellular miRNAs and their pathological and physiological significance are still poorly understood.Numerous studies have shown that miRNAs are important regulators of hepatic lipid metabolism regulation,and multiple miRNAs play key roles in NAFLD-related metabolic disorders.Mitochondria are one of the most abundant organelles in hepatocytes.Mitochondria contain a large number of miRNAs encoded by the nucleus,which can enter the cytoplasm from mitochondria and regulate their nucleus-encoded target genes and downstream molecules and thus cellular functions.For example,miR-29,which is mainly distributed in mitochondria,can regulate lipid metabolism in hepatocytes by repressing the expression of target genes PPARGC1A and ABHD5 in the cytosol.Therefore,we hypothesized that the abnormal mitochondrial-cytoplasmic distribution of miRNAs may play an important role in the development of NAFLD.However,nothing is known about what abnormalities in the distribution of miRNAs between mitochondria and cytoplasm in NAFLD,and what roles and mechanisms these abnormalities have in hepatic lipid metabolism and NAFLD pathogenesis.Objectives:1.Identification of miRNAs with significantly altered mitochondrial-cytoplasmic distribution in NAFLD pathogenesis.2.To reveal the role of mitochondrial-cytoplasmic redistribution of miRNAs in abnormal liver lipid metabolism in NAFLD and its mechanism by using high abundance miRNAs with the most significant changes in mitochondrial-cytoplasmic distribution.Methods:1.Animal grouping and modelingFour-week-old male SD rats,randomly divided into two groups,were fed normal and highfat chow(51%fat)for 12 weeks for modeling.During this process,ultrasound examination of rat liver was performed and rats and their livers were weighed.Hepatic index was calculated.The serum levels of triglycerides(TG),total cholesterol(TC),alanine transaminase(ALT)and aspartate transaminase(AST)were measured in each group of rats using an automated biochemical analyzer.AST)levels.2.Liver sample collection and mitochondrial purificationAfter 12 weeks of feeding,rats were executed,livers were quickly removed,clipped and liver tissue was resuspended and ground using pre-chilled RSB hypotonic buffer,and purified mitochondria were obtained after centrifugation using differential centrifugation.3.Purification and sequencing of miRNAsThe miRNAs from liver cells and their mitochondria were isolated and purified according to the instructions of the miRNAs Isolation and Purification Kit,and then libraries were created using the illumine TruSeq Small RNA Library Prep Kit and miRNAs were sequenced using the SE50 model.4.miRNAs sequencing data analysisFirst,the raw sequencing data were processed with fastp to obtain clean data,and the clean data were compared with rat genome using bowtie software,and the hit reads were compared with miRNAs sequence database to obtain the annotation results and expression matrix of miRNAs.Then Deseq2 was used to compare the expression differences of liver and mitochondrial miRNAs between normal and NAFLD rats.The target genes of typical miRNAs with abnormal mitochondrial-cytoplasmic distribution were predicted using mirDIP4.1,and the target gene jointly predicted by three unused software was selected for functional enrichment analysis and regulatory network analysis,while the core sub-network analysis was performed using the MCODE plugin of Cytoscape 3.9.1.5.Cell culture and processingComplete medium was used to culture HepG2 and HEK293T cells.The HepG cells2 were randomly grouped and stimulated with different concentrations of oleic acid(OA)and palmitic acid(PA)for 24h or 48h,respectively,to construct cellular models of NAFLD.HEK293T cells were mainly used for dual luciferase experiments.6.Co-transfection of hepatocytes with expression vectors of miRNAs and their target genes/downstream signaling moleculesAfter HepG2 cells were grown to 60%fusion,mimics or suppressors of miRNAs were transfected with Opti-MEM medium after proportional dilution of RNAiMAX and siRNA,and the fluid was changed after 6 hours of transfection.Lipofectamine 3000 and expression vectors and P3000 were transfected with Opti-MEM medium at proportional dilutions of Lipofectamine 3000 and expression vectors of the corresponding target genes/downstream signaling molecules,respectively,and changed after 6 hours and incubated for another 36-48 hours.7.Double luciferase experimentThe pmirGLO luciferase vectors containing the corresponding miRNAs binding site sequences on the 3’UTR of the target gene and the corresponding mutant sequences were constructed,and then the constructed vectors were co-transfected with miRNAs mimics or negative control sequences in HEK293T cells,and the luciferase activity of each group of cells was detected after 48 hours.8.Detection of lipid metabolism-related indicators and key signaling molecules in hepatocytesThe qPCR experiments were performed according to the instructions of the qPCR kit to detect the expression of each miRNAs and their target genes and downstream lipid metabolismrelated key genes at the mRNA level,and the expression were quantified absolutely using the standard curve method.HepG2 cells were lysed and total protein was extracted,and the expression of miRNAs target genes and downstream key genes related to lipid metabolism at the protein level was detected using Western blotting.Western blotting results were measured and analyzed by optical densitometry using ImageJ software.Hepatocytes and liver tissues were fixed with 4%paraformaldehyde and then stained with Oil Red O solution to observe lipid deposition in cells and tissues under the microscope.9.Statistical analysisStatistical analysis was performed with GraphPad Prism 9.0 and the R package.The Shapiro-Wilk test was used to determine whether the data conformed to a normal distribution.Continuous numerical data conforming to a normal distribution were expressed as mean±standard deviation(x ± s),and two groups were compared using a two-tailed t-test,and a oneway ANOVA was used for comparison between multiple groups.Non-normally distributed numerical data were compared between two groups using the Wilcoxon rank sum test and between multiple groups using the Kruskal-Wallis test.p<0.05 or adjusted p<0.05 indicates statistical significance.All experiments were repeated independently at least three times.Results:1.High-fat diet induces NAFLD in SD ratsHigh-fat diet significantly increased the body weight of SD rats,caused significant accumulation of liver lipids,increased liver index and significantly increased serum triglyceride levels,and induced the development of NAFLD in rats.2.The levels of multiple miRNAs in the liver of NAFLD rats were significantly altered in the mitochondria,but their expression levels in the whole hepatocytes were unchangedSequencing of miRNAs showed that 49 miRNAs in the livers of NAFLD rats had no significant change in expression levels in whole cells compared with rats in normal controls,but their relative mitochondrial abundance(Rm/t,the ratio of the expression levels of miRNAs in mitochondria to their expression levels in whole cells)was significantly altered,in other words,their mitochondrial-cytoplasmic distribution was significantly abnormal.Among these miRNAs,48 miRNAs showed a significant decrease in Rm/t,i.e.,a significant translocation to the cytosol;only miR-33-3p showed a significant increase in Rm/t,i.e.,a significant translocation to the mitochondria.On this basis,we further validated the miRNAs with the most significant change in mitochondrial-cytoplasmic distribution using absolute quantitative PCR for the fraction of miRNAs.The results showed that miR-30e-3p,miR-30c-2-3p,miR-339-5p,miR-194-3p,miR-125a-5p,miR-125b-5p and miR-92a-3p,seven miRNAs,had significantly decreased distribution in mitochondria,i.e.,significantly translocated to the cytosol;while miR33-3p in mitochondria distribution was significantly increased,i.e.,significantly translocated toward the mitochondria.3.Potential target genes of miRNAs that are clearly transduced from mitochondria to cytoplasm are closely associated with hepatic lipid metabolismThe mirDIP4.1 database was used to predict potential target genes of miRNAs with unchanged total expression in NAFLD rat hepatocytes but significantly decreased levels in mitochondria,i.e.,significantly translocated from mitochondria to cytoplasm,and to screen the target genes predicted by three different software together,followed by functional enrichment analysis of these potential target genes.The results showed that the potential target genes of these miRNAs with significantly altered mitochondrial-cytoplasmic distribution were significantly enriched for several important biological processes and signaling pathways related to liver lipid metabolism,such as lipid biosynthesis,metabolism,storage,SREBP signaling pathway and MAPK signaling pathway.4.Four miRNAs with significant changes in mitochondrial-cytoplasmic distribution(miR30e-3p,miR-30c-2-3p,miR-339-5p and miR-194-3p)target and inhibit the expression of TBL1XR1,PDK2,OGT and ACLY in hepatocytes,respectivelyTo further explore the role and mechanism of mitochondrial-cytoplasmic redistribution of miRNAs in the development of NAFLD,we focused on miR-30e-3p,miR-30c-2-3p,miR-3395p and miR-194-3p with the most significant changes in mitochondrial-cytoplasmic distribution at high abundance,and investigated their effects on lipid metabolism in hepatocytes and their mechanisms.The results showed that palmitic acid and oleic acid significantly down-regulated the levels of miR-30e-3p,miR-30c-2-3p,miR-339-5p and miR-194-3p in the mitochondria of HepG2 cells,while significantly increasing their levels in the cytoplasm,but the total expression of these miRNAs in the cells was not significantly altered,i.e.,these four miRNAs underwent a mitochondria to cytoplasm with significant translocation/redistribution.By qPCR,Western blotting and dual luciferase assays,we demonstrated that miR-30e-3p,miR-30c-2-3p,miR-3395p and miR-194-3p target and repress the expression of cytosolic-encoded TBL1XR1,PDK2,OGT and ACLY genes,respectively,in HepG2 cells.5.miR-30e-3p,miR-30c-2-3p,miR-339-5p and miR-194-3p promote lipid accumulation in hepatocytes by synergistically upregulating the expression of SREBP1-C through its target genesTo further clarify the role of miR-30e-3p,miR-30c-2-3p,miR-339-5p and miR-194-3p in abnormal liver lipid metabolism,we co-transfected HepG2 cells with their analogues and/or expression vectors of their target genes,respectively.The changes of lipid accumulation in the cells with different treatments were then detected under free fatty acid(FFA)stimulation conditions using Oil Red O staining.The results showed that the above four miRNAs couldpromote lipid accumulation in HepG2 cells through their target genes.It has been suggested that the target genes of the above four miRNAs,TBL1XR1,PDK2,OGT and ACLY,can regulate the expression of SREBP 1-C,a gene that plays a key role in lipid metabolism.Moreover,our results also showed that the expression of SREBP1-C was significantly increased in the liver of NAFLD rats.Accordingly,we conducted further functional studies,and the results showed that the above four miRNAs could indeed significantly upregulate the expression of SREBP1-C in hepatocytes through their target genes and promote lipid accumulation in hepatocytes.Conclusions:1.There are several miRNAs in the liver of NAFLD rats with unchanged total expression but significantly altered distribution between mitochondria and cell plasma,and these miRNAs are closely related to the lipid metabolism function of hepatocytes.2.High abundance of miR-30e-3p,miR-30c-2-3p,miR-339-5p and miR-194-3p,the most significantly altered mitochondrial-cytoplasmic distribution in the liver of NAFLD rats,were significantly translocated to the cytoplasm and targeted to repress the expression of the nucleus-encoded TBL1XR1,PDK2,OGT and ACLY genes,respectively.3.miR-30e-3p,miR-30c-2-3p,miR-339-5p and miR-194-3p synergistically upregulate the expression of SREBP1-C through their target genes,thereby promoting lipid accumulation in hepatocytes.