Effects Of N-3 Unsaturated Fatty Acids On The Regulation Of MiRNA In Exosomes And The Inhibition Of Obesity In Mice

Objective: Using fat-1 transgenic mice that can convert n-6 polyunsaturated fatty acids(n-6 PUFAs)into n-3 polyunsaturated fatty acids(n-3 PUFAs),to study the effect of increasing n-3 PUFAs on the body weight and the expression of exosomal mi RNAs in the blood of mice,then explore the mechanism of n-3PUFAs inhibiting obesity through exosomal mi RNAs.Methods: Select the six-week-old healthy littermate mice,extract DNA from mice tail and identify fat-1 gene by PCR and agarose gel electrophoresis.The identified mice were divided into four groups: the fat-1 normal diet(ND)group,the wild normal diet group,the fat-1 high-fat diet group(HFD)and the wild high-fat diet group.Six mice in each group were fed for 8 weeks.Then measure body length and body weight,and calculate Lee’s index of mice to determine whether the model was successfully established.After successful establishment of the model,extract and identify exosomes in peripheral blood of mice;exosomes were extracted from plasma by high-speed centrifugation;the appearance of exosomes was observed by electron microscopy;the size of exosome particles was detected by particle size technology;and the exosomes were identified by WB tracing.Extract and isolate RNA from exosomes,and construct libraries and perform high-throughput sequencing of mi RNAs.According to the sequencing results,find the target genes and related signaling pathways by KEGG enrichment analysis,and make the scatter plot of related pathways.Select the higher differentially expressed mi RNAs,and then refer to Target Scan,miRDB and mi TarBase three databases to find out the target genes and pathways with higher importance and relevance,then make the mi RNA-gene interaction map,and find the miRNA-target gene interaction relationship.To validate the association of mi RNAs with obesity and the role they play in obesity.Results: The obesity model of mice was established successfully.The body weight of fat-1 transgenic mice was significantly lower than that of wild-type mice;The body weight of fat-1 transgenic mice fed with HFD was significantlylower than that of the wild-type mice fed with HFD.the exosomes and total RNA in the blood of mice in each group were successfully extracted,and the exosomes were identified successfully.The content and quality of total RNA met the sequencing requirements and can be used for high-throughput sequencing.Quantitative analysis of high-throughput sequence showed that there were 46 differentially expressed miRNAs(p < 0.05 and FC value ≠ 1)among different mouse groups,including 29 with high expression and 17 with low expression.KEGG functional pathway analysis revealed that the mi RNAs and target genes were related to Endocytosis pathway,which was related to lipid metabolism.There were 38 target genes and 31 related mi RNAs in the pathway.Among all the differentially expressed miRNAs,21 miRNAs were most significantly differentially expressed(p < 0.001 and FC value > 1.5).They were found to be closely related to lipid metabolism-related pathways after pathway analysis.6 of the important miRNAs(mmu-miR-665-3p,mmu-miR-122-5p,mmu-miR-122-3p,mmu-miR-194-5p,mmu-miR-34c-5p,mmu-miR-223-3p)were predicted to fall at key positions of unsaturated fatty acid metabolism pathways,and there were 9 corresponding target genes located on this pathway(Fads1,Elovl2,Elov6,Hadha,Scad1,Scad2,Hsd17b12,Acot2,Acot4).Meanwhile,12 target genes(Arf6,H2-T-ps,Arrb1,Ist1,H2-T10,Wwp1,Snx4,IL2 rb,Mvb12b,Rab11,fip3,Kif5 a,Nedd4l)corresponding to the six predicted mi RNAs were located in the endocytic pathway.These target genes are also closely related to lipid metabolism and obesity.Conclusion: The increasing of n-3 PUFAs in mice can effectively reduce the weight of mice and inhibit obesity.n-3 PUFAs can regulate the expression of mi RNAs in exosome,among which the significantly different expressed mi RNAs are related to obesity,and their related target genes are effectively focused on the regulation of fatty acid metabolism and other molecular pathways related to lipid metabolism.The experimental results suggest that this may be one of the mechanisms by which n-3 PUFAs reduce body weight and inhibit obesity,and also suggest that the regulation of mi RNA may be a potential strategy for treatment of obesity in the future.