Selection Of Differential Expressed MiRNA And Study On The Expession And Regulation Of MiRNA-33 During Goose Fatty Liver Formation

MicroRNAs(miRNAs)are 21-23 nt non-coding RNA molecules that can regulate their target gene expression post-transcriptionally.Previous researches have established that miRNAs play important roles in the liver lipodystrophies in human and mouse.While goose has a natural ability to store lipid in its liver,without any obvious pathological symptoms,which provides a good model for studies on lipid metabolism in liver.In this study,next-generation sequencing technologies will be used to detect the differentially expressed miRNAs and genes during the process of liver fattening,then the miRNA target genes will be predicted using some bioinformatics methods,and combined with previous studies the regulatory network will be constructed between the miRNAs and the genes to screen the miRNAs and the target genes playing a central role.Isolation and culture of gosse primary hepatocytes,real-time PCR analysis and western blot analysis will be used to verify some of the target genes and the regulation of the expressed miRNAs(miRNA-33 and CROT),so as to further clarify the mechanism of goose fatty liver formation.The main results are as follows:1.The effect ofoverfeeding and quality of small RNA sequencing.Our study showed that,compared to control geese,both the body weight(BW)and liver weight(LW)of the overfed geese were greater at day 19 of overfeeding(P<0.05).To be specific,the BW of overfed geese and control geese were 7.57±0.48 kg and 3.87±0.25 kg(n=6),respectively;the LW were 826.26±182.26 g and 80.63±12.80 g,respectively and the ratio of LW to BW were 10.86%and 2.08%,respectively.In line with the drastic increase of liver weight in the overfed geese,the liver color turned from a normal liver color(deep red)to a fatty liver color(khaki).These findings suggested the overfeeding experiment was successful and severe steatosis was induced in the livers of the overfed geese.The quality analysis of RNA samples for sequencing showed that the concentration of all samples were greater than 80 ng/?l,and there was no degradation and pollution,indicating the high quality of RNA samples.2.Analysis of differentially expressed miRNA(DEM)and regulation network construction.We sequenced the liver RNA samples from overfed geese and control geese at day 7,14 and 19 days of overfeeding.After filtering,splicing and annotating of the raw data,1163 miRNAs with annotation were obtained.Totally,30(17 upregulated),48(36 upregulated)and 151 DEM(114 upregulated)were identified at day 7,14 and 19 of overfeeding,respectively.Combined with previous transcriptome sequencing results,234,445 and 845 target genes were predicted at the corresponding time points,with 156,270 and 211 upregulated target genes,respectively.Pathway analysis of the predicted target genes indicated that the carbohydrate,lipid and amino acid metabolism,the cell growth and deathand the immune diseases pathway were the main enriched pathways.Based on the results of target gene prediction,the relationship among the target genes were obtained from STRING database and then the miRNA-mRNA regulation network were constructed.3.The expression and regulation of miRNA-33 and its target gene-CROT.In our previous study,we found that miRNA-33 was related to lipid metabolism and CROT was preliminarily proved as its target gene.In this study,we also found that miRNA-33 was significantly induced by overfeeding at different stages(especially at the latter stage).Thus,we performed further study on the expression and regulation of miRNA-33 and its target gene-CROT.Real-time PCR results indicated that miRNA-33 was indeed induced by overfeeding in the liver of Landes geese.In addition,the expression of miRNA-33 was also induced in abdominal fat and breast muscle,indicating that miRNA-33 did play a role in lipid metabolism.Further study showed that CROT(the predicted target gene of miRNA-33)was suppressed by overfeeding in the liver of Landes geese,at both mRNA and protein level.The expression pattern of CROT was just the opposite of miRNA-33,suggesting their potential regulative relationship.In vitro study indicated that CROT mRNA was significantly suppressed in goose primary hepatocytes treated with miRNA-33 mimics or miRNA-33 plasmid while induced when treated with miRNA-33 inhibitor,which suggested that CROT was the target gene of miRNA-33.We further treated the goose primary hepatocytes with glucose and insulin,and found that glucose could significantly suppress the expression of miRNA-33 while induce the expression of CROT.However,insulin treatment significantly induced miRNA-33 while suppressed CROT,which are the same with the expression pattern in liver.These results not only suggested the regulative relationship between miRNA-33 and CROT,but also indicated that insulin resistence of goose liver induced by overfeeding was the main reason for miRNA-33 upregulation,which further suppressed CROT and participated in goose liver formation.