| MiRNAs have been reported to be important regulators for lipid metabolism. MiR-200family is closely related to the lipid metabolism. For miR-200family, its regulation on genes associated with milk fat synthesis in goat mammary gland has just been uncovered, however, the expression profile and the function of miR-200family in goat mammary gland have not yet been studied. To investigate the role of miRNAs in mammary gland on milk composition, we establish the expression profiles of miR-200family in mammary gland of Xinong Saanen dairy goats, analyse the correlation between miR-200family members expression and milk composition contents, and measured the mRNA expression of miR-200predicted targets as miR-200down-regulated in goat mammary epithelial cells(GMECs). We also used goat specific miR-200a-inhibitor to suppress endogenous miR-200a expression in GMECs, and investigated the effect of miR-200a expression inhibition on cell growth, lipid droplet formation, as well as the mRNA expression of genes involved in milk lipid metabolism, providing a basis for the fuction validation of miR-200.The major results of this paper are as follows:(1) By using the Real-Time qPCR, miR-200family (miR-200a, miR-200b, miR-200c, miR-141, miR-429) was identified to be expressed in goat mammary gland tissue. Their expression levels during mid-lactation were all significantly higher than that during dry period (P<0.01). Furthermore,223bp pri-miR-200a,241bp pri-miR-200b,389bp pri-miR-200c,409bp pri-miR-141, and293bp pri-miR-429were identified. The sequence homology of these pri-miRNAs with bovine is96%,99%,96%,96%,98%, respectively. Additionally, two miRNA clusters were cloned from DNA, including2211bp miR-200b-200a-429and1011bp miR-200c-141. Their sequence homology with bovine is,94%and95%.(2) Correlation analysis showed the significant correlations between miR-200a and miR-141(R=0.720, P<0.05), and miR-200b and miR-200c (R=0.957, P<0.01). And the expression levels of miR-200c significantly correlated with the contents of goat milk protein, SNF, and FPD (P<0.05). By use software of the bioinformatics prediction and analysis, we found STAT4, a key regulator for milk protein and fat synthesis, is a predicted target gene of miR-200a and miR-141. Suppression of miR-200a expression in GMECs can up-regulate the expression JAK、STAT4、STAT5A、STAT5B associated with JAK-STAT signaling pathways.(3) Expression of miR-200a and mRNA related to milk fatty acid metabolism was measured in negative-miRNA-inhibitor-transfected GMEC (control) and miR-200a-inhibitor-transfected GMEC at48h post transfection. In comparision with control, the endogenous miR-200a expression in miR-200a-inhibitor-transfected GMEC decreased by66%(efficiency of interference is66%); and the expression of FASN(+3.82, P<0.01), ACACA(+1.97, P<0.05)、ACSS1(+1.74,P>0.05)、SCD (+1.77,P>0.05)、ACC1(+1.75, P>0.05)、ATGL (+1.85, P<0.05)、 LPL (+24.87, P<0.01)、ADRP (+8.23, P<0.01)、 TIP47(+1.43, P>0.05)、PPARy (+96.72, P<0.01)、PGC (+4.36, P<0.05) all increased. Furthermroe, suppression of miR-200a expression increased the number of lipid droplets, affected cells growth, the content of triglyceride and total cholesterol was not significant (P>0.05).In conclusion, this study domanstrates important roles of miR-200a in fatty acid metabolism, lipid droplets and cell growth in GMECs. This study also indicates a novel biological role of the miR-200family in regulating protein and lipid metabolism of milk. These results provide a basis for the fuction validation of miR-200a in goat mammary gland and are helpful with understanding the theory of lipid metabolism in goat mammary gland tissues. |
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