Screening And Function Analysis Of The Key Mirna In Milk Lipid Metabolism Of Mammary Tissues Of Dairy Goats

Comparing with the cow milk,goat milk(milk of Capra hircus)includes a higher level of unsaturated fatty acids,along with total fat,vitamins,calcium,carbohydrates,and proteins.Milk fat metabolism is a complex procedure controlled by several factors.Mi RNA regulate expression of genes and influence a series of biological procedures,such as fatty acid metabolism.Here we screened expression of goat mammary gland’s mi RNA during early-lactation,peak-lactation,late-lactation,non-lactating(“dry”)then sequenced GMECs(goat mammary gland epithelial cells)using prolactin treatment and found 6 mi RNA expresses remarkably.We also study about the functional,regulation relationship and molecular mechanism role of screening mi RNA.The results as follows: 1.Screening and analysis of key fat metabolism mi RNA in different lactation periodsTo reveal the connection between the fatty acid,milk fat metabolism and the mi RNA regulation,here we screened the mi RNA expression including early-lactation,peak-lactation,late-lactation,non-lactating(“dry”),including 793 Bos taurus primary mi RNA and 267 Capra hircus primary mi RNA from mi RBase.In the screening,all the mi RNA with p-value under 0.05 and four-fold change were chosen as candidate mi RNA.Then we chose two important factors related to launching and sustaining lactation that were PRL(2.5 μg/ml),to treat GMES as well as a second round of screening.It was found that mi R-30e-5p,mi R-15 a,mi R-181 b,mi R-148 a,mi R-17-5p and mi R-135 b were of significant difference both in PRL.2.MiR-181b Suppresses TAG via target IRS2 and regulating in the Hippo pathwayWe illustrated that the over-expression of mi R-181 b impaired lipid metabolism while the knockdown of mi R-181 b promoted lipid metabolism in GMEC.Using Luciferase reporter assay and Western Blot,IRS2 was illustrated to be a mi R-181b’s potential target gene.What is interesting is that mi R-181 b regulates multiple key components in the Hippo pathway,such as LATS1 and YAP1 in GMECs.In conclusion,our findings indicated that mi R-181 b suppress lipid metabolism by means of regulating multiple genes in the Hippo pathway and target IRS2.3.MiR-30e-5p and miR-15a synergistically regulate fat metabolism via LRP6 and YAP1Utilizing a Luciferase reporter assay and Western Blot,Yes associated protein 1(Yap1)and LRP6,were demonstrated to be targets of mi R-148 a and mi R-17-5p in goat mammary epithelial cells(GMECs).We also verified that mi R-30e-5p regulated β-catenin,which in turn modulated YAP1,and mi R-30e-5p repressed the expression of YAP1 during lipid metabolism in GMECs.Moreover,we demonstrated that the over expression of mi R-30e-5p and mi R-15 a promoted lipid metabolism while the knockdown of mi R-30e-5p and mi R-15 a impaired lipid metabolism in GMECs.These findings extended the discovery of mi R-30e-5p and mi R-15 a functioning in mediating adipocyte differentiation by suggesting its role in promoting milk fat synthesis.4.Mechanism of prolacting inhibition of mi R-135 b via methylationIn this study,we found out prolactin(PRL)was significantly down-regulated the expression of mi R-135 b through quantitative PCR analysis,which was functionally related to lipid metabolism via LATS2.We also found out PRL depends on processing time and concentration when relates to downward expression of mi R-135 b.In addition,there is a Cp G island in the mi R-135 b of 5’-flanking region in research about the regulation of the expression of mi R-135 b,the Cp G island will inhibit the expression of mi R-135 b by methylated.Based on previous research,we further studied the function and mechanism of mi R-135 b in the PRL stimulating GMECs.Expression of DNMT I(DNA methyl transferase I)was increased in GMECs with the PRL stimulation,which led to DNA methylation in mi R-135b’s Cp G island of the 5’-flanking region and inhibited the transcription and expression of mi R-135 b.At the same time,down-regulation of mi R-135 b led to rise of large tumor suppressor 2(Lats2)expression,proliferation of mammary gland and metabolism of milk fat,which eventually leading to the formation of GMECs lactation.5.MiR-148 a and mi R-17-5p synergistically regulate milk TAG synthesis via PPARGC1 A and PPARA in goat mammary epithelial cellsThe research indicated that mi R-148 a,mi R-17-5p,PPARGC1A(PGC1a)and PPARA are highly expressed in the goat mammary gland in early-lactation and non-lactation.Utilizing a Luciferase reporter assay and Western Blot,PPARA,an important regulator of fatty acid oxidation,and PPARGC1 A,a major regulator of fat metabolism,were demonstrated to be targets of mi R-148 a and mi R-17-5p in goat mammary epithelial cells.It was also revealed that mi R-148 a expression can regulate PPARA,and mi R-17-5p represses PPARGC1 A in GMECs.Furthermore,the over expression of mi R-148 a and mi R-17-5p promoted triacylglycerol(TAG)synthesis while the knockdown of mi R-148 a and mi R-17-5p impaired TAG synthesis in GMEC.These findings extend the discovery of mi R-148 a and mi R-17-5p as important components that promote TAG synthesis.In conclusion,our findings indicate that mi R-148 a cooperates with mi R-17-5p to regulate fatty acid by repressing PPARGC1 A and PPARA in GMECs,which promotes further study on the function of mi RNA in lipid metabolism in ruminant mammary cells.In conclusion,we screened expression of goat mammary gland’s mi RNA,and found that 6 mi RNA expresses remarkably.The research was also determined together with the correlation between mi RNA expression and fatty acid composition in goat milk to explore the molecular mechanism of synergistic mi RNA regulating the fatty acid metabolism of dairy goats,which will provide theoretical and experimental basis for manipulating goat milk fatty acids content,and understanding the goaty flavor formation etc.