Role Of MiR-696Targeting PGC-1α In Hepatic Gluconeogenesis Of Ob/ob Mice

Background:MicroRNAs (miRNAs) are a class of endogenous and noncoding RNA molecules of19-23nt in length, which regulate gene expression at the posttranscriptional and translational levels. PGC-la plays an important role in regulating gluconeogenesis and fatty acid oxidation in liver. In the present study, we explored the mechanism of miR-696in regulating PGC-la in vivo and vitro and the possible role in the development of hepatic gluconeogenesis.Methods:In liver of ob/ob mice and C57BL/6mice, we examined PGC-la protein level by western blotting analysis and miR-696expression level by qRT-PCR. We infected mouse primary hepatocytes with pre-miR-696-LV, pre-NC-LV, anti-miR-696-LV, anti-NC-LV and then examined the efficiency of infection at72hours under fluorescent microscopy. QRT-PCR and western blotting analysis were then applied to examine the level of miR-696and PGC-la protein in mouse primary hepatocytes at72hours after infection. Next, we conducted luciferase binding assays using a psiCHECK-2luciferase plasmid to determine whether miR-696suppressed PGC-la through direct binding to its3’-UTR. At last we examined the expression of PEPCK as an indicator to test the downstream biological consequences of miR-696-driven inhibition of PGC-1α expression through western blotting analysis.Results:(1) We found that the expression level of PGC-la protein was significantly increased in the liver of ob/ob mice,2.15times that of normal C57BL/6mice; while, miR-696level in liver of ob/ob mice was downregulated significantly, only0.21times that of normal control. The results suggest that miR-696may regulate PGC-la expression negatively in mouse liver.(2) The efficiency of infection at72hours screened by GFP (green fluorescent protein) signal was higher than80%. The level of miR-696was increased to6.7folds in primary hepatocytes infected by pre-miR-696-LV compared controls; whereas, miR-696level was reduced to59%after primary hepatocytes were infected by the anti-miR-696-LV compared with the anti-NC-LV. The expression of PGC-1α was decreased to41%in miR-696over-expressed hepatocytes and enhanced by87%in miR-696knock-down hepatocytes. These results prove that miR-696negatively regulates PGC-1α in primary hepatocytes.(3) The overexpression of miR-696resulted in a62%reduction of firefly luciferase reporter activity; while the knockdown of miR-696resulted in a17%increment of firefly luciferase reporter activity. These results unequivocally demonstrate that miR-696directly recognizes the3’-UTR of the PGC-la transcript.(4) Western blotting analysis showed that the level of PEPCK protein was increased to47%in the liver of ob/ob mice compared with normal C57BL/6mice. At72hours after overexpression or knockdown of miR-696, the expression level of PEPCK was significantly inhibited by treatment with pre-miR-696-LV, only0.37times that of normal control; and significantly elevated by treatment with anti-miR-696-LV,1.51times that of normal control.Conclusions:MiR-696directly recognizes the3’-UTR of the PGC-la transcript and negatively regulates PGC-la protein expression in mouse liver; and then coordinates the expression of characteristic hepatic gluconeogenic enzymes, including PEPCK through coactivating transcription factors, such as HNF-4a, FOXO1; and therefore plays an important role in the development of hepatic gluconeogenesis and insulin resistance.