Screening And Expression Analysis Of MiRNAs Involved In Regulation Of Casein Synthesis By Arginine

Functional amino acids-Arg can promote the casein synthesis in mammary epithelial cells to some extent, in previous studies, our group analyzed and researched the related factors and functional genes that involved in the casein synthetic pathway in the mammary epithelial cells.. On this basis, this experiment will further explore the effect of arginine on the expression of miRNA in bovine mammary gland and its molecular mechanism. The research included the following four parts.Exp.1 Prediction of miRNA Regulating Casein Synthesis in Arginine Metabolic PathwayThe objective of this study was to forecast and screen the target miRNAs by two network database called Pictar and Targetscan. we started with the miRNA that regulate and control the related functional genes in arginine regulating casein synthesis metabolic pathway. The results showed that, through comparing the predicted results from two databases, the mmu-miR-325, mmu-miR-3062, mmu-miR-471-3, etc.53 miRNAs in total which related to casein protein synthesis in arginine metabolic pathway were selected as candidate miRNAs.Exp.2 The study of Important miRNAs Regulating Casein Synthesis in Arginine Metabolic Pathway in vitroThe 2nd generation bovine mammary epithelial cells that reserved in our laboratory were cultured in vitro by adding againine in this experiment to assay the selected important miRNAs in Exp.1. The treatments were designed as follows, Arg× 1 group (278.00 mg/L), Arg×2 group (556.00 mg/L) as experimental group respectively, and Arg× 0 group (0.00 mg/L) was set as the control group. Then we used RT-PCR to detect the expression of related candidate miRNAs in arginine regulating casein synthesis pathway. Results showed that the mmu-miR-743a, mmu-miR-543, mmu-miR-101α, mmu-miR-760-3p, mmu-miR-1954, mmu-miR-712, mmu-miR-574-5p, mmu-miR-468 and mmu-miR-875-3p had significant differences between Arginine group and Control group. This suggests those 9 candidate miRNAs may have regulating effect in the arginine metabolic pathway and then influence casein synthesis of in mammary epithelial cells.Exp.3 Effects of Arginine Infusion on Casein Synthesis and miRNAs Expression in Bovine Mammary GlandSix Holstein cows were chose, which have similar body weight, parity (4), lactation period (80+2 D), milk yield (21.0+1.0 kg) and body condition (3.0) as experimental animals, they were randomly divided into 3 groups, respectively were the control group, Arginine group and Alanine group, every group had 2 test cows, in a 3 x 3 Latin square trail. This study was conducted to investigate the effects of arginine infusion through jugular vein on casein synthesis and miRNAs expression in bovine mammary gland. The results showed that infusion of arginine was beneficial to improve the milk protein content, milk quality and the synthesis of casein. The expression of CSN1S1, CSN1S2 were significantly increased. Moreover, the expression of mmu-miR-743a, mmu-miR-543, mmu-miR-760-3p, mmu-miR-1954, mmu-miR-712, mmu-miR-574-5p that chose from the in vitro trail all had high level expression (P<0.05); but the miR-468 had a low level expression (P<0.05) in Arginine group. This indicated that Arginine may regulate related genes in synthetic pathway through influencing those 8 miRNAs expression and finally control the synthesis of casein.Exp.4 Effects of Jugular Vein Infusion of Arginine on Nitrogen Metabolism and Immune Function in Lactating CowsThis study was conducted to investigate the effects of arginine infusion through jugular vein on nutrient digestion, nitrogen utilization and immune performance of lactating cows. The experimental design was the same as the third experiment. The results showed that milk protein nitrogen yield in arginine group was significantly higher than those in the other 2 groups (P< 0.05); the arginine group also had the highest ratio of milk protein nitrogen to nitrogen intake (P>0.05); the arginine group significantly increased the serum immunoglobulin IgG, the plasma total protein and the ratio of albumin and globulin (P<0.05); but the glutamic-pyruvic transaminase and glutamic oxalacetic transaminase were not affected by perfusion; meanwhile IL-1 and TNF-α were also high ein this group. In conclusion, perfusing arginine in lactating cows can improve the milk protein synthesis through improving the nitrogen digestion and utilization rates, and perfusing arginine can also increase the total protein concentration, proportion of albumin, immunoglobulin protein IgG and cytokines IL-1 and TNF-a in cow blood, and finally improve the body immunity of lactating cows and fetal immunity passive.