Insulin induced mTOR expression, glucose consumption and lactate production via miR-99a.

<p>(A) The luciferase reporters containing the wild type and mutant 3′-UTR of mTOR at predicted miR-99a binding site were constructed and verified by sequencing. HEK-293 cells were transfected with wild type or mutant reporter plasmid, scrambled control miRNA (miR-SCR) or miR-99a precursor, and pRL-TK plasmid, and the relative luciferase activity was calculated as the ratio of firefly/renilla activities and normalized to that of the control. Data were presented by mean±SE (n = 3). **, indicates significant decrease compared to the control (p<0.01). (B) Cells were transfected with miR-99a or miR-SCR at 40 nM. After 72 h, the expression of mTOR and GAPDH was determined by immunoblotting (up panel). Relative quantitative levels of mTOR were measured as above (bottom panel). * and **, indicate significant decrease compared to the miR-SCR group (p<0.05 and p<0.01). (C) HepG2 and HL7702 cells were transfected as above, then starved and treated with or without insulin. Cell pellets were collected and the relative density of mTOR expression was determined. The data were analyzed by ANOVA. *, indicates significant increase compared to the control without insulin treatment (p<0.05); ^# and ^##, indicate significant decrease compared to miR-SCR and insulin treatment (p<0.05 and p<0.01). The glucose consumption (D) and lactate production (E) were tested in supernatant of treated cells as above. * and **, indicate significant increase compared to the control without insulin treatment (p<0.05 and p<0.01); ^##, indicates significant decrease compared to miR-SCR and insulin treatment (p<0.01).