Gene Expression Analysis in NCI-H520 human cancer cells upon deadenylase silencing

The first and rate-limiting step in eukaryotic mRNA decay is the shortening of the poly(A) tail catalyzed by a family of enzymes known as deadenylases. In humans, several deadenylases have been recognized so far, yet it is not clear what the advantage is to have many enzymes catalyzing the same reaction. It is hypothesized that specific deadenylases may target unique subsets of mRNAs, or multiple deadenylases can act on the same mRNA, with discrete but overlapping functions. To understand the biological significance of the diversity of these enzymes we silenced the expression of several deadenylases, including PARN, NOC, CNOT6, CNOT6L, and CNOT7, in human cells of cancer origin (NCI-H520; squamous lung cancer), and analyze the impact on gene expression with microarrays. The selected deadenylases were silenced with sequence-specific short hairpin RNAs delivered in NCI-H520 cells using cationic liposomes. Wild type cells, as well as cells transfected with empty vector or shRNA with no homology to known gene sequences, were used as controls and data normalization. Puromycin selection was performed 12h post transfection. Cells were harvested 72h upon transfection, RNA was extracted and gene expression was analyzed.