Analysis Of Hepatic Gene And MiRNAs Expression Profiles In The Piglet Fed Diet With Different Protein Level And Functional Validation Of Key MiRNAs

The utilization efficiency of nitrogen in pigs was affected by many factors,and dietary protein level was the key factor affecting the growth and nutrient utilization efficiency of swine.Liver is an important metabolic organ of mammalan,and plays an important role in biochemical processes such as amino acid metabolism,protein synthesis,urea cycle,glycolipid metabolism and so on.The urea cycle is the main form of excessive nitrogen emission in vivo,but its molecular regulation mechanism is not clear until now.In this study,we measured ptrotein and mi RNA expression in hepatic tissue of pigs fed low protein diets using high-throughput sequencing to obtain a comprehensive view to reveal their functions in urea metabolism and specifically to identify critical mi RNAs that play key roles in the regulation of urea cycle,and to provide a theoretical basis for the molecular mechanism of urea cycle regulation by mi RNA.Experiment 1 To investigate the effects of low protein diets on the hepatic proteome profile,weaned 28-d old piglets were randomly fed one of the two diets based on corn-soybean: a normal protein diet(NCP)and a low protein diet(LCP)for 30 d.Liver and blood samples were collected for proteomic and subsequent analyses.The proteomic analysis revealed that a total of 6222 proteins were identified in the liver of the three test groups,172 proteins were differentially expressed between the 17%CP and 20%CP groups,while 234 different proteins in 14%CP compared with 20%CP.The KEGG pathway analysis showed these proteins were involved in carbohydrate,protein,lipid,amino acid,and vitamin metabolism,oxidative stress,immune function and ECM pathways.Western blotting analyses and detection of other relevant indicators revealed that decreased hepatic protein and urea synthesis;while 14% CP significantly inhibited protein synthesis,but 17% CP promoted the liver protein synthesis,Furthermore,liver gluconeogenesis,and fatty acid ?-oxidationis apparently reduced and is associated with increased glycogenesis and lipogenesis.Experiment 2 To assess the mi RNA expression profile in a low protein diet and identify mi RNAs involved in the regulation of the hepatic urea cycle,in present experiment,the mi RNA expression profiles of liver in different protein diets assessed by Solexa sequencing,The results showed a total of 606 mi RNAs were detected,and there were 52 differentially expressed mi RNAs between the low protein group and normal groups,with 30 up-regulated and 22 down-regulated.Expression of three up-regulated mi RNAs(ssc-mi R-19 b,ssc-mi R-19 a hsa-mi R-192-3p?R-1)in 14% CP group were opposite to that in 17% CP group.Bioinformatics analysis showed that 15 of these differentially expressed mi RNAs targeted to key enzyme of urea cycle and their regulatory factors.Experiment 3 In this study,we selected eight mi RNAs which were transfected into porcine primary hepatocytes by using their synthesize mimics to observe the changes in extracellular urea concertration,The results indicated that three mi RNAs potentially targeting SIRT5(ssc-mi R-19 b,ssc-mi R-145-5p,ssc-mi R-204)significantly affected urea production of cell compared with the NC group,and CPS1 protein expresion were significantly reduced(P<0.05).Inhibitor of ssc-mi R-19 b and ssc-mi R-204 rescued this reduction.Then we used the dual fluorescein plum reporting system to verify the target of these three mi RNAs.The luciferase activity of the wild-type SIRT5 reporter was significantly reduced(P < 0.05)by ssc-mi R-19 b mimic compared with the negative control.The reduction was rescued both by mutation and deletion of the seed sequence.To futher confirm ssc-mi R-19 b regulates urea synthesis by targeting SIRT5,porcine primary hepatocytes were treated with different concentrations of amino acids.The results showed that high concentrations of amino acids increased the synthesis of urea and the protein expression of SIRT5 and CPS1(P <0.05).Then,the contents of urea and SIRT5 and CPS1 protein expression were further inhibited after AA treatment with mi R-19 b mimics intervening.Results further confirmed that low protein diets decreased the urea production by promoting the expression of ssc-mi R-19 b and thus reducing SIRT5 protein expression.Experiment 4 To explore the role of mi R-204 s in urea synthesis GR was predicted bioinformatically to be a target of mi R-204,followed by validation by using dual luciferase reporter system.Then,the synthetic ssc-mi R-204 mimic or inhibitor was transfected to primary hepatocytes,with NC as negative control.The expression of GR and CPS1 protein and urea content were detected.The results showed mi R-204 mimic significantly inhibited the urea synthesis,GR and CPS1 protein expression(P<0.05),while the inhibitor has opposite effect.In addition,the contents of urea and GR and CPS1 protein expression was further inhibited after AA treatment with mi R-204 mimics intervening.These above results confirm that ssc-mi R-204 inhibits urea synthesis by targeting GR,in the case of low protein diets.In this study,we revealed hepatic proteomic and mi RNAs expression changes in swine fed normal and low protein diets,and demonstrated that the mi R-19 b and mi R-204 negatively regulated urea synthesis by targeting SIRT5 and GR respectively,which are a positive regulator of CPS1,the rate limiting enzyme in the urea cycle.