Post-transcriptional Regulation Of PPAR-alpha By Micro RNA In Fatty Acid Metabolism

liver has a central role in energy metabolism, hepatic energy metabolism is tightly controlled by Multiple nutrient, hormonal, substrates and circadian rhythm synergistically, have been identified to regulate glucose, lipid, and amino acid metabolism in the liver. It acts as a hub to metabolically connect to various tissues, including skeletal muscle and adipose tissue, and contributes to energy metabolism balance. Associated with fed and fasting, liver acts as a sensor of energy requirement, manipulates glucose and lipid anabolic metabolism and catabolism or store as fuel. Energy metabolism change significantly during fed and fasting circumstances. In the fed state, the liver predisposes to glycogen synthase and triglyceride synthesis, as well as energy transportation. And during fasting, glycolysis, gluconeogenesis, fatty acid beta oxidation and ketogenisis significantly increased. PPARα is highly expressed in active FA metabolism tissues, including liver, muscle, brown adipose tissue, heart and kidney. As a member of PPAR family, PPARα is a ligand-activated transcription factor, plays a critical role in lipids and carbohydrates metabolic regulation. Numerous reports have showed that PPARα serve as a key positive regulator of FAO and ketogenisis during fasting state. The expression of PPARα significantly increased in this circumstance. CPT-1α and ACADM are rate-limiting enzymes of FAO. CPT-1α catalysis the entrance of long-chain FA-Co A into mitochondrial which is the first stap of FAO. ACADM mediates straight middle chain FA(between C4-C12) FAO in mitochondrial. HMGCS2 is rate-limiting enzyme of ketogenisis. All of the three enzymes are direct downstream targets of PPARα and the expression of three enzymes are transcriptional positively regulated by PPARα. However major efforts have devoted in the posttranslational modification of PPARα, including acetylation, phosphorylation, polyubiquitination and glycosylation et.al. There’re few reports to on the mechanism ofthe gene expression level changes. In our study,we focus on the posttranscriptional level regulation of PPARa by mi RNA. From the bioinformatics prediction on the 3’UTR gene sequence of PPARα, among the 10 potential mi RNAs candidates, 4 mi RNAs level changes were found to be inversely related with PPARa increases in the fasting liver examples of mice. Further experiments at cellular levels provide some intitial evidences of direct regulations on PPARa by the 4 mi RNAs, such as mi R-21,mi R-27 a,mi R-106 b,mi R-181 a.Objectives:[1] To observe the alteration of the expression of PPARα and micro RNA during fasting state and screening candidate micro RNA responsible for PPARα regulation in vivo and in vitro.[2] To explore the role of candidate micro RNAs in hepatic metabolism.[3] To validate the regulation of PPARα by specific micro RNA.Methods and Results:[1] In the liver of mice, the accumulation of lipid droplet significantly increased after 24 h fasting and decreased to normal level after refed. q RT-PCR results showed that the m RNA level of PPARα and the downstream targets CPT-1α,HMGCS2,ACADM were significantly increased during fasting and then recovered to normal after refed. while mi R-21,mi R-27 a,mi R-106 b,mi R-181 a were down-regulated. Indicating the role of these micro RNAs.[2] second part, we employed micro RNA mimics to validate the function of these micro RNAs in Hep G2 cell line. During low glucose treatment, micro RNA mimics significantly inhibited the increase of PPARα and the downstream targets expression at both m RNA and protein levels,suggesting that mi R-21,mi R-27 a,mi R-106 b,mi R-181 a participated in the hepatic lipid metabolism in a PPARα dependent manner.[3] At the last part, we found that inhibited mi R-21,mi R-27 a,mi R-106 b,mi R-181 a by micro RNA inhibitor, there’s a significant increase of PPARα and the downstream targets m RNAs. Then we employed luciferase activity assay and validated these micro RNA directly regulated PPARα expression in a posttranscriptional manner.Conclusions: We focus on the posttranscriptional regulation of PPARα, supporting the novel roles of mi R-21,mi R-27 a,mi R-106 b,mi R-181 a in hepatic lipid metabolism during fasting via regulating PPARα expression. q RT-PCR and western blot results showed dynamic changes of hepatic PPARα expression during fed and fasting states, these micro RNAs displayed the opposite expression trends and they coulddirectly regulate t PPARα m RNA expression via recognizing the 3’UTR binding sites.