Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor activity

Abstract

The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary MEF senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity.

Figure 1. Characterization of MEF under 6T15 propagation regimen.

(A) Proliferation curve of MEF expressed as cell doublings per passage. (B) Quantification of p19ARF, p16 and p21 transcripts per passage (each point represent one passage) normalized to that of MEF at passage 0. (C) p21 protein level at the various passages. The value reported under each lane represents the average of two independent experiments.

Figure 2. MEF immortalization is characterized by a p53-dependent events.

DNA content profile per cell at p1/p5 (A), p5/p6 (B) and p6/p10 (C). (D) Fold change of miR-34a and p53 transcripts at various passages normalized to that of MEF at passage 0. (E) p53 protein level at the various passages. The value reported under each lane represents the average of two independent experiments.

Figure 3. The re-expression of miRNAs down regulated in immortalized MEF reduces cell proliferation.

(A) Quantification of miR-20a, miR-21, miR-28, miR-290 and miR-34a per passage normalized to that of MEF at passage 0. Dashed lines indicate the transition from passage 5 to 6. (B) Cell cycle phase distribution (%) of MEF from p1 to p5. (C) Population cell doubling of immortalized MEF after transfection of miR-NC, miR-20a, miR-21, miR-28, miR-290 and miR-34a. Each bar represents the mean ± SD of three biological replicates (*=p<0.05). (D) Expression of ASF/SF2 protein in primary (p3 and p5) and immortalized (p10) MEF. The value reported under each lane represents the average of two independent experiments. (E) Expression of ASF/SF2 after the transfection of immortalized MEF with either miR-NC or miR-28. The value reported under each lane represents the average of two independent experiments.