AMPK-related MicroRNAs Expression Profile In Mouse Hepatocytes And Function Research

Objective:Adenosine monophosphate-activated protein kinase (AMPK) is the core for cell to adapt to the changes in different environment and nutrition. Liver is the most important organ to storage and release carbohydrate. MicroRNAs (miRNAs) are a newly discovered group of small molecular RNA, which play a role in almost all of the life processes including energy metabolism. This research was intended to screen miRNAs which might be involved in AMPK signaling pathway, to look for miRNAs which participate in regulate energy metabolism in AMPK pathway, to predict their target genes and possible biological function with bioinformatics, and to further validate their role in the regulation of gluconeogenesis in AMPK signaling pathway.Method:1. Screening of hepatocyte AMPK related miRNAs:Male C57BL/6mouse primary hepatocytes were cultured in vitro. Dibutyryl cyclic adenosine monophosphate (Bt2-cAMP) was added into medium as Bt2-cAMP group, and the experimental groups were added different concentrations (0.125mM,0.25mM,0.5mM,1mM) of5-aminoimidazole-4-amide-l-B-D-ribofuranoside (AICAR). AMPK, cyclic adenosine monophosphate-response element binding protein regulates transcription coactivator2(CRTC2) protein expression and their phosphorylation status were detected by western blotting. Peroxisome proliferator-activated receptor coactivator1a (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6pase) genes expression were detected by real time PCR. Proper concentration of AICAR was chosen for miRNAs microarray and Bt2-cAMP group was as control.2. Bioinformatics analysis of AMPK related miRNAs:Upregulated and downregulated miRNAs’target genes were predicted by Targetscan website. The relationship between two groups of targets and diseases were analysed by FunDo analysis. MiRNAs which fold changes significantly in AICAR group and abundance in liver were chosen for further research. Targets were predicted by multiple websites and evaluated miRNAs’function in the regulating process of glucose metabolism in AMPK pathway.3. AMPK regulated gluconeogenesis through miR-29:The expression of miR-29 family and p85a, PEPCK, G6pase genes were detected by real time PCR. MiR-29family minics was constructed and transfection into mouse primary hepatocytes. After transfection successful, the expression of p85a, PEPCK and G6pase genes were detected by real time PCR, the expression of p85a protein was detected by western blotting. Further research was focus on the expression of miR-29family, p85gene and its protein in liver of fasting14h mice.Result:1. Screening of hepatocyte AMPK related miRNAs:The expression of AMPK and CRTC2phosphorylation were inhibited by Bt2-cAMP intervention in primary mouse hepatocytes, and the expression of PGC-la, PEPCK and G6pase genes were increased (P<0.01). AICAR intervention promoted the phosphorylation of AMPK and CRTC2, inhibited the expression of PGC-1a, PEPCK and G6pase (P<0.01). According to the result, AICAR0.5mM was chosen for the microarray experiment. There were41miRNAs significantly altered in AICAR-treated sample (fold change:>2) compared with untreated control sample. Among them,19miRNAs were upregulated.2. Bioinformatics analysis of AMPK related miRNAs:According to the results of microarray, target genes were prediction by Targetscan. The number of total targets predicted for13upregulated miRNAs and20downregulated miRNAs were2456and3090, respectively. FunDo analysis showed that32diseases were found to be statistically enriched with the upregulated miRNAs targets, whereas46diseases were found with downregulated miRNAs targets. The top5liver related diseases were cancer, diabetes, hypertension, obesity and heart failure (P<0.01). Targets were predicted by multiple websites. It comes to that pik3rl was target gene of miR-29family.3. AMPK regulated gluconeogenesis through miR-29:After AICAR intervention, the expression of the three family members of miR-29decreased significantly than those in control group, p85a gene expression increased (P<0.01), and PEPCK, G6pase genes expression decreased (P<0.01). The miR-29family members were highly homologous, and the3family members in the hepatocytes were close to the level and change trend. After the miR-29family minics was constructed and successful transfected into mouse primary hepatocytes, p85a gene and protein expression decreased (P<0.05), and PEPCK, G6pase genes expression increased (P<0.01). The expression of miR-29family of fasting mice higher than that of control group, and the expression of p85a gene and protein was decreased (P<0.01).Conclusion:1. AICAR activated AMPK and its downstream signaling pathway, inhibited the expression of gluconeogenesis related genes in mouse primary hepatocytes. AICAR influenced the expression of mouse primary hepatocytes miRNAs spectrum. There were a variety of miRNAs altered significantly, which may be involved in the regulation of AMPK signaling pathway.2. Bioinformatics analysis showed the expression of miRNAs under AICAR intervention may be related to pathogenesis of cancer and metabolic diseases.3. AICAR influenced the expression of miR-29family in mouse hepatocytes. p85a, as the target gene of miR-29, may involve in the regulation of gluconeogenesis in AMPK’s pathway. AMPK signaling pathway played a role in the regulation of gluconeogenesis partly by miR-29and its target gene p85a.