MicroRNA Expression Profiles In The Fibroblast Cells Derived From In Vivo Fertilization, Cloned And Transgenic Cloned Cattle

The control metabolic homeostasis is very important to maintain the body’s normal physiological function and health. Some important transcription factor may directly or indirectly influence nutritional and metabolic chain, such as cholesterol, lipid, glucose, and insulin can rapidly change the program of gene expression and thus maintain homeostasis. miRNAs is important regulatory factors of the metabolic process, through with transcriptional regulation mechanisms by cover or cross to regulate metabolic balance. miRNAs may also be involved in the regulation of somatic cell cloned, transgenic cloned animals in epigenetic.The miRNAs expression profiles of bovine fibroblasts which derived from two in vivo fertilized (Ferti), two somatic cell nuclear transfer (SCNT) and two fat-1 transgenic (TG) cattle were obtained through Solexa sequencing technology. Therefore, six miRNAs libraries were constructed and their expression patterns were compared by Mireap software, and target genes regulated by high abundance differentially expressed miRNAs were predicted. Then the biological pathways of these target genes were studied by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis, to reveal the relationships between miRNAs differential expression and target genes in those cattle.The results were as follows:1. Three hundred and sixty-seven known miRNAs were identified, in which 83,19 and 23 were specifically expressed in Ferti, SCNT and TG cattle, respectively, while 14 were expressed in all cattle. Meanwhile,178 novel miRNAs were found, in which 17,1 and 4 were specifically expressed in Ferti, SCNT and TG cattle, respectively, while 1 were expressed in all cattle.2. Among those differentially expressed miRNAs, there are a series of up-regulated and down-regulated miRNAs in fibroblasts cells, the abundantly differentially expressed top ten miRNA were detected, validated and compared by quantitative real-time PCR in both Ferti group /SCNT group and Ferti group/TG group, meanwhile, high expression of two novel miRNA were validated by qPCR validation and prediction partially of novel miRNA structure. GO and KEGG pathway analysis revealed differential expression of miRNA target genes significantly enriched in the Metabolic pathway, MAPK signaling pathway, Calcium signaling pathway, Purine metabolism pathway, Ubiquitin-mediated proteolysis pathway, Pyrimidine metabolism pathway and Cell cycle pathways.3. Combined with previous studies and closely related genes in fat metabolism, through bioinformatics analysis, FGF10, LPL and ATGL were predicted to be the target genes of bta-miR-21. In the fibroblasts of Ferti cattle, the bta-miR-21 mRNA expression level was significantly higher than that in the SCNT and TG cattle, while the mRNA expression levels of FGF10 and ATGL were significantly lower than the other two, which may indicate that bta-miR-21 played a negative regulation role to FGF10 and ATGL. Preliminary consider that these target genes may be involved in fat regulation and metabolic processes.4. Functional identification of microRNA:overexpression experiment was carried out by transfecting fibroblasts with bta-miR-21 mRNA. The results confirmed the negative regulation role of bta-miR-21 to FGF10 and ATGL and indicated that FGF10 and ATGL were the target genes of bta-miR-21. Thus, the bta-miR-21 may affect the lipid metabolism through the regulation of FGF10 and ATGL.In conclusion:1. In the study, the miRNA library of fibroblast were established by using different embryos (in vivo fertilization, clone and fat-1 transgenic cattle) from the same genetic background. The miRNA information involved in the offspring development for clone and transgenic clone cattle were obtained, which supplied important data for study the dysplastic mechanism in clone and transgenic clone embryos.2. The regulation role of bta-miR-21 and its target genes (FGF10 and ATGL) were detected in the fibroblast of in vivo fertilization, clone and fat-1 transgenic cattle, which implied that bta-miR-21 and its target genes (FGF10 and ATGL) might be the important targets for the development regulation in clone and transgenic clone embryos.