Reversible, interrelated mRNA and miRNA expression patterns in the transcriptome of Rasless fibroblasts: functional and mechanistic implications

BACKGROUND: 4-Hydroxy-tamoxifen (4OHT) triggers Cre-mediated K-Ras removal in [H-Ras(-/-);N-Ras(-/-);K-Ras(lox/lox);RERT(ert/ert)] fibroblasts, generating growth-arrested “Rasless” MEFs which are able to recover their proliferative ability after ectopic expression of Ras oncoproteins or constitutively active BRAF or MEK1. RESULTS: Comparison of the transcriptional profiles of Rasless fibroblasts with those of MEFs lacking only H-Ras and N-Ras identified a series of differentially expressed mRNAs and microRNAs specifically linked to the disappearance of K-Ras from these cells. The rescue of cell cycle progression in Rasless cells by activated BRAF or MEK1 resulted in the reversal of most such transcriptional mRNA and microRNA alterations. Functional analysis of the differentially expressed mRNAs uncovered a significant enrichment in the components of pathways regulating cell division, DNA/RNA processing and response to DNA damage. Consistent with G1/S blockade, Rasless cells displayed repression of a series of cell cycle-related genes, including Cyclins, Cyclin-dependent kinases, Myc and E2F transcription targets, and upregulation of Cyclin-dependent kinase inhibitors. The profile of differentially expressed microRNAs included a specific set of oncomiR families and clusters (repressed miR-17 ~ 92, miR-106a ~ 363, miR-106b ~ 25, miR-212 ~ 132, miR-183 ~ 182, and upregulated miR-335) known for their ability to target a specific set of cellular regulators and checkpoint sensors (including Rb, E2F and Cdkns) able to modulate the interplay between the pro- and anti-proliferative or stress-response pathways that are reversibly altered in Rasless cells. CONCLUSIONS: Our data suggest that the reversible proliferation phenotype of Rasless cells is the pleiotropic result of interplay among distinct pro- and anti-proliferative, and stress-response pathways modulated by a regulatory circuitry constituted by a specific set of differentially expressed mRNAs and microRNAs and preferentially targeting two cross-talking signalling axes: Myc-Rb-E2F-dependent and Cdkns-p53-dependent pathways.