Effect of miR-122 overexpression of mRNA distribution on the polyribosomes

The aim of this study was to identify at genome-wide level, the effect of miR-122 on the distribution of its responsive genes on the polyribosomes. For this purpose human hepatoma cells Huh-7 were transfected for 48 hrs with LNA-based miR-122 inhibitors (122-PM) or miR-122 mimics (122-MIM). Following the treatment with cycloheximide, cells were lysed and layered on a 10-50% sucrose gradient. Following ultracetrifigation the polyribosomes were separated in 12 different fractions The individual polyribosomal fractions of cells transfected with either miR-122 mimics (122-MIM) or antagomiRs (122-PM) were then combined to generate polyribosomal pools of decreasing densities [i.e., P1 = Heavy (fractions A1-A4), P2 = Middle (fractions A5-A8), and P3 = Light (fractions A9-A12)]. These six different RNA pools (i.e., three densities for two conditions for two different experiments) were hybridized to a total of 12 Affymetrix microarrays.