Study On The Identification Of MicroRNAs In Bovine Mammary Epithelial Cells And The Screening Of MicroRNAs Related To Milk Fat Metabolism

To determine how the synthesis or metabolism of lipid in milk is regulated at the mi RNA level, small RNA libraries were constructed from each of the primary mammary epithelial cell(p MEC) cultures derived from Chinese Holstein dairy cows that produced extreme differences in milk fat percentage. Solexa sequencing and bioinformatics analysis were then used to determine the abundance of mi RNAs and their differential expression patterns between two p MECs. Differentially expressed mi RNAs and their potential functions were subsequently predicted by GO and KEGG annotation. Finally,mi RNA and their reverse-complementary target gene candidates that were annotated to the pathway of fatty acids metabolism were screened and identified by the methods of mi RNA over expression, mi RNA inhibition, 3′-UTR Dual luciferase reporter gene analysis,real-time q PCR and Western Blot experiments.The main results are as followed:1. Six Chinese Holstein cows with the highest and lowest milk fat percentage(n= 3 for each group) were selected. And two different mammary gland epithelial cells were cultured by the method of tissue nubbles culture and were named as p MEC-HH(cells cultured from mammary gland with highest milk fat ratio) and p MEC-LL(cells cultured from mammary gland with lowest milk fat ratio) respectively. Then the identification results of cell morphology, cytogenetics, growth and secretory characteristics showed that two different p MECs have the normal phenotype and biological characteristics. TAG content comparison results showed the significant differences between p MEC-HH and p MEC-LL which indicated the sustained functionality of milk fat synthesis and secretion in p MEC cultures.2. Mi RNAs from p MEC-HH and p MEC-LL were sequenced and identified subsequently by Solexa sequencing technique. Totally, 292 known mi RNAs were identified with 251 mi RNAs appeared in both libraries. However, 17 mi RNA/mi RNA* were detected only in p MEC-HH small RNA library and 21 mi RNA/mi RNA* were specifically expressed in p MEC-LL. Also,116 novel bovine mi RNAs were detected. 32 novel mi RNAs were expressed in both libraries;60 novel mi RNAs were specifically expressed in p MEC-HH; 84 novel mi RNAs were specifically expressed in p MEC-LL.3. The comparison results of mi RNA expression patterns between the two p MEC libraries showed that 97 known mi RNAs were differentially expressed at significant level( p < 0.05) while 91 known mi RNAs were co-expressed in both libraries. Among these, 38 conserved mi RNAs were up regulated and 59 conserved mi RNAs were down regulated in p MEC-LL compared with those in p MEC-HH. Further more, a total of 9987 genes were predicted as putative target genes for 97 differentially expressed mi RNAs. GO and KEGG pathway annotation for the predicted target genes showed that 305 biological processes with p < 1 were annotated. And the pathways related to the lipid metabolism were focused. 61 target genes were annotated to the pathway of fatty acid metabolic; 13 target genes were annotated to the pathway of fatty acid biosynthesis; 32 target genes were annotated to the pathway of fatty acid elongation; 28 target genes were annotated to the pathway of unsaturated fatty acid biosynthesis.4. 13 known mi RNAs were selected randomly and analyzed by the stem-loop real-time q PCR at both cellular and tissular levels. The results confirmed the similar mi RNA expression patterns between the cells and fresh tissues. Furthermore,a high degree of consistency was also observed between the results of q PCR and Solexa sequencing data. All above results indicated that screening for mi RNAs associated with milk fat synthesis at the cellular level was sensitive and feasible.5. By the validation of Real-time q PCR and Western Blot analysis,four bovine conserved mi RNAs that related to fatty acid metabolism in mammary gland were screened through combining to their predicted target genes. They are bta-mi R-33 a,predicted target gene ELOVL5 and ELOVL6; bta-mi R-21*, predicted target gene ELOVL5 and PTGIS;bta-mi R-152,predicted target gene PTGS,PRKAA1 and UCP3;bta-mi R-224,predicted target gene LPL,ALOX15 and GST.6. The target gene of bta-mi R-152 and their functions were further identifiedusing the Dual luciferase reporter gene analysis. The results showed that bta-mi R-152 influenced the total TAG content in p MEC by target-binding the candidate gene UCP3. In addition, the cell viability showed no significant difference before and after the transfection. Therefore, the results suggested that the function of bta-mi R-152 could be crucial for milk fat synthesis in bovine mammary epithelial cells.Two types of bovine mammary gland epithelial cells differentiated in milk fat synthesis were constructed successfully. The mi RNA expression pattern of the two p MECs were also significantly different. Four mi RNAs which related to milk fat metabolism and regulation were finally screened. They are bta-mi R-33 a, bta-mi R-21*,bta-mi R-152 and bta-mi R-224. Further functional and target gene identification results showed that bta-mi R-152 can affect the milk fat content by target-binding the candidate gene UCP3.