Figure 1. miR-122 is downregulated in c-Myc-induced liver tumors.

A. Northern blot analysis of precursor and mature miR-122 levels in the normal liver (L) from FBVN and (Myc-off) mice, as well as benign liver (N) and tumor (T) from the same (Myc-on) mouse. Total RNA (25 µg) from each sample was separated by denaturing PAGE (15% acrylamide), transferred onto Nylon membrane, cross-linked and subjected to Northern blot analysis with ³²P-labeled anti-miR-122 deoxyoligonucleotide. Membrane was exposed to 4hr and 72hr at −80°C to detect mature and precursor miR-122, respectively. The blot was hybridized to anti-sense 5S rRNA probe to demonstrate equal loading of RNA. B. qRT-PCR and Western blot analysis of c-Myc expression in the liver (Myc-off), Myc-on benign liver (N) and tumor (T) tissues in the presence and absence of doxycycline, respectively. Whole tissue extracts (100 µg protein) were immunoblotted with an antibody specific for human c-Myc and reprobed with Gapdh antibody. C. qRT-PCR analysis of primary and mature miR-122 in Myc-off livers and Myc-on tumors using Taqman kit. Data was normalized to RNU6B. D. qRT-PCR analysis of mRNA levels of specified miR-122 targets in livers and tumors. C: control liver, T: tumor. E. Western blot analysis of selected miR-122 targets in liver and tumor whole tissue extracts (100 µg). L: FBVN liver, N: benign liver and T: Tumor. F. Luciferase reporter assay demonstrating Pkm2 is a target of miR-122. Upper panel: miR-122 cognate site in 3’-UTR of Pkm2 gene. Lower panel: relative luciferase activity in Hepa cells transfected with the psiCHECK2 harboring wild type (Pkm2-3’-UTR) or mutant (Pkm2-3’-UTR-mut) site along with miR-122 mimic (miR-122) or negative control RNA (NC).