| Fatty liver syndrome is a metabolic disease,which is common in laying hens with an incidence rate of 5%-30%.The mortality rate of fatty liver is low,but it will lead to the reduction of fertilization and hatching rate of breeding eggs,and affect the economic benefits of poultry breeding industry.Fatty liver is caused by the excessive accumulation of fat in the liver due to multiple factors such as increased liver fat synthesis,decreased oxidation and lipoprotein transport capacity.The current researches mainly focuse on the etiology,prevention and treatment measures,but the mechanism of fatty liver is less explored.In recent years,with the development of epigenetics,exploring the regulatory mechanism of miRNA in liver lipid metabolism has become a new research strategy.In the early stage,the group induced Jingxing-Huang(JXH)chicken to obtained fatty liver model by feeding high-fat and low protein diet,then bred their offsprings.miRNA sequencing was performed on the liver tissues of F1 generation fatty liver group and control group,and the sequencing results were jointly analyzed with the transcriptome sequencing,the differentially expressed miR-375 and its target genes recombination signal binding protein for immunoglobulin kappa J region(RBPJ)and mitogen-activated protein kinase kinase kinase 1(MAP3K1)were screened,then based on the verification of its target relationship,the molecular mechanism of miR-375 regulating chicken liver lipid deposition by targeting RBPJ and MAP3K1 was analyzed at the cell and population level,providing a theoretical basis for revealing the pathogenesis of chicken fatty liver and disease resistance breeding.The main results of this paper are as follows:1.Bioinformatics analysis of differentially expressed(DE)miRNAsAccording to the results of miRNA sequencing from the control and experimental group in the F1 generation of JXH chicken,we obtained 26 DE miRNAs,including 14up-regulated and 12 down-regulated miRNAs.GO and KEGG enrichment analysis showed that the target genes of DE miRNA are mainly related to molecular functions such as fatty-acyl-COA binding and carbohydrate derivative binding.In addition to participating in basic signal pathways such as MAPK and Notch signal pathway,it is also closely related to lipid metabolism pathways such as fatty acid elongation and citrate cycle(TCA cycle),suggesting that the above pathways may play an important role in regulating liver lipid metabolism.2.Verification of the targetable relationship between miR-375 and RBPJ and MAP3K1We screened the highest expressed miRNA(miR-375)according to the miRNA sequencing results for subsequent research.Predicting the target genes of miR-375 by Targetscan,and conjoint analysis of the transcriptome sequencing results of the group,then the target genes RBPJ and MAP3K1 were screened.According to the binding site,the vector containing RBPJ-3’UTR,MAP3K1-3’UTR and mutant 3’UTR sequence were constructed.The results of luciferase assay showed that miR-375 could specifically bind with RBPJ-3’UTR and MAP3K1-3’UTR,significantly reducing luciferase activity(P<0.01).To confirm the correlation between miR-375 and candidate target genes,after over-expressing miR-375 in chicken primary embryo hepatocyte,the expressions of RBPJ and MAP3K1 were significantly down-regulated(P<0.01).Reducing the expression of miR-375,the expressions of RBPJ and MAP3K1 were significantly upregulated(P<0.01).Otherwise,the results of vivo experiments show that miR-375 was significantly up-regulated in fatty liver group from F0-F3 generations(P<0.01;P<0.001),the expression trend of RBPJ and MAP3K1 was opposite to that of miR-375(P<0.01;P<0.001).Indicating that miR-375 can affect the m RNA levels of RBPJ and MAP3K1 by targeting RBPJ-3’UTR and MAP3K1-3’UTR specifically.3.miR-375 regulates lipid deposition in chicken embryo hepatocyte by targeting RBPJ and MAP3K1To further explore the mechanism of miR-375 and target genes RBPJ,MAP3K1 and fatty liver formation,chicken embryo hepatocyte was used as experimental materials to detect triglyceride content and observe lipid droplet deposition.The results showed that the content of triglyceride increased significantly(P<0.05)and lipid droplets increased after miR-375 mimic transfected into cells.When miR-375 inhibitor transfected,the trend of triglyceride(P<0.01)and lipid droplets in cells was opposite to the above situation,it shows that the increase of miR-375 will promote the deposition of lipids in hepatocyte.The study of its target gene found that after interfering with RBPJ,triglyceride content in cells increased significantly(P<0.05)and lipid droplet deposition increased.Moreover,the contents of triglyceride(P<0.01)and lipid droplets in cells decreased after over-expressing RBPJ.When the cells were transfected with si MAP3K1,the content of triglyceride increased significantly(P<0.05)and lipid droplets increased.In the rescue experiment,it was found that after co-transfection of si MAP3K1 with miR-375 inhibitor,compared with the miR-375 inhibitor group,the contents of triglyceride(P<0.001)and lipid droplets in cells were up-regulated after co-transfecting with si MAP3K1 and miR-375 inhibitor,it showed that si MAP3K1 changed the downregulation trend of miR-375 inhibitor on lipid deposition in cells.In addition,after cotransfecting cells with si MAP3K1 and miR-375 mimic,the trend of increased triglyceride content(P<0.05)and lipid droplet deposition were more significant.The above results showed that miR-375 regulated the lipid deposition of chicken embryo hepatocyte in vitro by targeting RBPJ and MAP3K1.4.Population validation of miR-375 targeting RBPJ and MAP3K1 regulating lipid metabolism of liver in chickenWhite Leghorn(WL)chicken were taken as the research object,individuals with normal and naturally occurring fatty liver were taken as the experimental materials,based on the analysis of liver phenotype and serum indexes,the expression levels of miR-375,RBPJ and MAP3K1 in the liver of WL chicken fatty liver group and non-fatty liver group were detected.The results showed that the expression of miR-375 was significantly up-regulated in liver of fatty liver group from WL chicken(P<0.001),and the expression of RBPJ and MAP3K1 was significantly down-regulated in fatty liver group of WL chicken(P<0.01;P<0.001).This result confirms that miR-375 and its target genes RBPJ and MAP3K1 have the same effect in regulating naturally or inductively occurring fatty liver in chicken.In conclusion,the results of this study clarify that miR-375 can act on chicken hepatocyte by targeting RBPJ and MAP3K1,and then have an effect on liver lipid metabolism.The results provide a basis for revealing the pathogenesis of chicken fatty liver and disease resistance breeding.In addition,chicken as a model animal,the study of the regulation mechanism of liver fat metabolism in chicken is also of reference significance for the study of human nonalcoholic fatty liver. |
PDF Full Text Request