The proto-oncogene LRF is under post-transcriptional control of MiR-20a: implications for senescence

Abstract

MicroRNAs (miRNAs) are short 20-22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3'UTR is demonstrated. To validate the interaction miR-20a/3'UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism, alternative to apoptosis; from this point of view the discovery of new physiological "senescence inducer" appears to be promising as this molecule could be used as anticancer drug.

Figure 1. miR-20a regulates LRF expression in MEF.

a: Sequences of miRNAs belonging to miR-17 family; b: RT-PCR analysis of pri-miRNA expression in MEF. Total RNA was extracted and amplified by RT-PCR using appropriate primers. The PCR products of ∼500 bp length are pri-miRNAs of the miR-17 family; c: Northern blot analysis of mature miR-20a expression in MEF. 20 µg of total RNA was analyzed with miR-20a probe or valine tRNA control probe; d, e, f: Interaction between 3′UTR of mmu-zbtb7a mRNA and miR-17 family. HEK293T cells were co-transfected with p-zbtb7a 3′UTR and increasing concentrations of p-miR-20a, p-miR-17 p-miR-106b or p-miR-26a control plasmid. 24 hours after transfection, cells were collected and the EGFP fluorescence intensity of each sample was determined with a FACscan analyzer. The relative expression of p-zbtb7a 3′UTR was obtained by the ratio of the mean fluorescence value of HEK293T cells transfected with p-miR-20a, p-miR-17 or p-miR-106b and the mean fluorescence value of HEK293T cells transfected with p-miR-26a control plasmid. Each bar represents the mean±SE of three independent experiments; g,: Transfection of p-miR-20a with either p-zbtb7a 3′UTR or p-zbtb7a 3′UTR_m in HEK293T. Cells were collected 24 hours after transfection and the EGFP fluorescence intensity of each sample was determined with a FACscan analyzer. The relative expression was obtained by the ratio of the mean fluorescence values of HEK293T cells cotransfected with p-miR-20a and either p-zbtb7a 3′UTR or p-zbtb7a 3′UTR_m normalized to that of HEK293T cells transfected with pEGFPC1. Each bar represents the mean±SE of three independent experiments. h i: Effects of over-expression/depletion of miR-20a on LRF expression.

Figure 2. Effects of miR-20a on LRF-p19ARF pathway.

a: q-RT-PCR of LRF, p19ARF and p21. Total RNA was extracted from MEF transfected with miR-20a, si-LRF or si-EGFP. mRNA level, detected by Real-Time PCR, was normalized to that of GAPDH. The values are the mean of two independent experiments. Western blot (b) and quantification (c) of LRF, p19ARF, p53 and p21. The band intensity was normalized to that of tubulin. Each bar represents the mean relative expression of LRF±SE of three independent experiments.

Figure 3. miR-20a induces senescence in MEF.

Detection of cell proliferation (a), SA-ß-gal positive (b) and binucleated (c) cells in wt MEF transfected with miR-20a. Detection of proliferation (d) and SA-ß-gal positive (e) cells in wt and LRF-null MEF retrovirally infected with PIG/miR-20a. Each bar represents the mean±SE of three independent experiments.

Figure 4. miR-20a regulates E2F1.

Western blot analysis showing the expression of LRF and E2F1 in wt (a) and LRF-null MEF (b) retrovirally infected with PIG/miR-20a; c: Western blot analysis showing the expression of E2F1 in MEF transfected with d-20a; d: Western blot analysis showing the expression of E2F1 and p19ARF in wt MEF transfected with si-LRF, si-E2F1, si-EGFP and miR-20a; e: percentage of SA-ß-gal positive cells in wt MEF transfected with si-LRF, si-E2F1, si-EGFP and miR-20a.

Figure 5. miR-20a regulates p16.

Western blot analysis showing the expression of p16 after miR-20a transfection in wt MEF (a,b) and in LRF-null MEF (c). Each bar represents the mean±SE of three independent experiments.