Effect of glucose availability on microRNA expression and Ago2 occupancy.

For cells to grow and proliferate both a growth signal and available nutrients are required. Several lines of evidence suggest that there is recriprocal regulation of nutrients by growth signals and growth signals by nutrients. Since microRNAs are regulated by growth signals, we sought to determine if microRNAs could also be regulated by nutrient availability. We found that several microRNAs change expression following glucose starvation and that their Ago2 occupancy remains constant. Immortalized Bax/Bak double-knockout mouse embryonic fibroblasts were deprived of glucose for 5 days and then re-fed glucose for 2 days. Cells were collected at the beginning of the starvation period (d0), following five days of glucose deprivation (d5), and following two days of glucose re-feed (d7). Total RNA was isolated from half of the collected cells using Trizol and the remaining cells were used to perform Ago2 immunoprecipitation followed by isolation of bound RNA.