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

Abstract

Background4-Hydroxy-tamoxifen (4OHT) triggers Cre-mediated K-Ras removal in [H-Ras-/-;N-Ras-/-;K-Raslox/lox;RERTert/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.ResultsComparison 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.ConclusionsOur 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.

Using miRNA-mRNA Interaction Analysis to Link Biologically Relevant miRNAs to Stem Cell Identity Testing for Next-Generation Culturing Development

“…Coincidentally, a publication reporting on miRNAs influencing proliferation and differentiation in C2C12 myoblasts showed a miRNA profile with considerable overlap with the differential MAPC/MSC profile [29], supporting our claim that the key MAPC and MSC miRNAs are involved in these processes. Furthermore, a study conducted in fibroblast also confirmed the pro-proliferative action of miR-17, miR-106a, miR-20a, and miR-18a and the antiproliferative action of miR-27b and miR-335 [74].…”

Concomitant deletion of HRAS and NRAS leads to pulmonary immaturity, respiratory failure and neonatal death in mice

“…Among RAS family members, only KRAS is essential for mouse development and viability whereas HRAS and NRAS are dispensable [12][13][14][15][16][17][18] . Transcriptomic analyses have identified specific transcriptional programs controlled by each RAS isoform 2 and suggested preferential involvement of HRAS with cell growth and proliferation, NRAS with immunomodulatory and apoptotic responses 19,20 , and KRAS with control of cell cycle progression 21,22 . Our earlier studies showed that HRAS/NRAS-DKO mice (expressing only KRAS) were viable and presented no obvious phenotypes, but significantly lower-than-expected numbers of adult DKO animals were obtained when breeding NRAS-KO (HRAS +/− ;NRAS −/− ) and HRAS-KO (HRAS −/− ; NRAS +/− ) mice kept on mixed genetic background 12 .…”

MEK Inhibitor Suppresses Expression of the miR-17-92 Cluster with G1-Phase Arrest in HT-29 Human Colon Cancer Cells and MIA PaCa-2 Pancreatic Cancer Cells

“…Recently, it was reported that PD0325901 suppressed expression of miR-25, which is a member of the miR-106b-25 cluster, a paralog of the miR-17-92 cluster (5), with upregulation of PTEN expression in human melanoma M14 cells harboring a BRAF mutation (19). On the other hand, it was also demonstrated that both the miR-17-92 and miR-106b-25 clusters were up-regulated by RAS/RAF signaling using 'Rasless' fibroblasts (20). These results suggest that activation of the mitogen-activated protein kinase pathway induces expression of oncogenic miRNA clusters, including the miR-17-92 and miR-106b-25 clusters, and vice versa, a MEK inhibitor might suppress expression of these oncogenic miRNA clusters.…”