MicroRNA-221-222 regulate the cell cycle in mast cells

Abstract

MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that have emerged as key posttranscriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development and differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells and found that miR-221-222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors, and potential cis-regulatory regions that control miR-221-222 transcription. Overexpression of miR-221-222 in a model mast cell line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221-222 partially counteracted expression of the cell-cycle inhibitor p27(kip1), we found that in the mouse alternative splicing results in two p27(kip1) mRNA isoforms that differ in their 3' untranslated region, only one of which is subject to miR-221-222 regulation. Additionally, transgenic expression of miR-221-222 from bacterial artificial chromosome clones in embryonic stem cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides further insights on miR-221-222 transcriptional regulation as well as evidences that miR-221-222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli.

Conflict of interest statement

Disclosures: The authors have no financial conflict of interest.

Figures

Figure 1. Increased levels of miR-221-222 in activated mast cells

a) Alignment of mature miR-221 and miR-222 sequences. The ‘seed’ sequence is shaded in gray. b) PipMaker (27) analysis of conservation of 3.18 kb of genomic sequence surrounding the miR-221 and miR-222 sequences, in five different species. Each dot represents sequence identity. Only regions that were at least 50% identical are plotted. c) Northern blot of 4-8 weeks differentiated murine BMMCs left resting or stimulated for 1h or 24h with PMA and ionomycin. D5 and D10 are respectively Th1 and Th2 clones, while naïve T CD4+ lymphocytes were isolated from spleen and lymph nodes of 6 weeks-old mice. d) TaqMan PCR for miR-221 expression in primary Th1, Th2 and BMMC cells. OT-II CD4+ lymphocytes were cultured for 6 days in Th1- and Th2-skewing conditions and were restimulated for 6h with PMA and ionomycin. Th1 cells were 98% IFNγ+ with no IL4+ cells as assessed by intracellular cytokine staining (not shown), while Th2 cells were 70% IL4+ and 2% IFNγ+. Four-weeks IL-3 differentiated BMMCs were either left resting or were stimulated for 6h with PMA and ionomycin. Total RNA was extracted and first-strand synthesis was performed with a miR-specific primer on 10ng of total RNA. TaqMan PCR was performed for miR-221 and snoRNA202 as endogenous control. e) Northern blot of differentiated BMMCs cultured for 1h in the presence of IgE only (3 μg/ml), followed by crosslinking with an anti-IgE Ab (2-6 μg/ml) for 24h. BMMCs were also stimulated for the same amount of time with PMA and ionomycin. In both c) and d) the same blot was stripped and reprobed for the indicated miRNAs, as well as for an Arg-tRNA as loading control.

Figure 2. MiR-221-222 are transcriptionally upregulated in activated mast cells

a) Time-course of stimulation with PMA and ionomycin of differentiated BMMCs. Ethidium bromide staining of the gel is shown as loading control. b) RT-PCR on RNA extracted from differentiated BMMCs stimulated for the indicated times with PMA and ionomycin. Actin mRNA was used as a loading control. The RT-PCR shown is representative of 3 independent experiments. A schematic representation (not to scale) of the primers used to detect the primary transcript is shown on top.

Figure 3. Transcriptional requirements for miR-221-222

a) Northern blot of 4-8 weeks differentiated BMMCs stimulated for 24h with PMA and ionomycin alone or in combination. Cells were also either left resting or were treated with CsA. The same blot was stripped and reprobed for an Arg-tRNA as loading control. Quantification of the Northern blot via PhosporImager is indicated by the numbers underneath the gel. b) Primary BMMCs were differentiated for 4 weeks from mice deleted for nfat1 and from control littermates, and were either left resting or were stimulated for 24h with ionomycin alone or in combination with PMA, with or without pre-treatment for 20min with CsA. Quantification of the Northern blot was performed with a PhosphorImager and it is shown in the graph. c) Northern blot of differentiated BMMCs pre-treated for 1h with an IKK-inhibitor or DMSO vehicle followed by PMA and ionomycin stimulation for 24h. The same blot was stripped and reprobed for the indicated miRNAs, as well as with an Arg-tRNA as loading control. The figure is representative of three independent experiments. d) Quantitative RT-PCR for miR-222 performed on BMMCs treated for 1h with an IKK-inhibitor or DMSO vehicle followed by PMA alone or PMA and ionomycin stimulation for 24h. U6 was used to normalize expression. Expression in the differently treated samples is compared to the DMSO treated sample, which is set to one.

Figure 4. DNase I HS pattern in the miR-221-222 locus

a) DNase HS analysis in unstimulated primary murine Th2 cells differentiated for 1 week and BMMC cells. The probe recognized a 17.9kb BamHI fragment encompassing the miR-221 and miR-222 sequences (shown in the schematic representation in panel d)). The black triangles indicate increasing amounts of DNaseI enzyme. b) The same blot from panel a) was stripped and reprobed with a probe recognizing the BamHI fragment farther upstream in the miR-221-222 locus (24.3kb in the schematic representation in panel d)). c) DNase HS analysis in primary murine BMMCs differentiated in vitro for 4-8 weeks and either left resting or stimulated for 24h with PMA and ionomycin. The probe used recognized the 17.7kb KpnI fragment shown in d). d) Schematic representation of all of the DNaseI HS sites identified in the 33 kb of genomic sequence upstream of miR-221 and miR-222. Identified sites are superimposed an analysis of sequence conservation performed in UCSC Genome Browser (http://genome.ucsc.edu/). Putative NF-κB and NFAT binding sites identified by bioinformatics analysis are also indicated.

Figure 5. A higher proportion of mast cells stably expressing miR-221-222 is in G1/G0

a) MC-9 cells transduced with miR-221-222 lentiviral vectors were sorted for GFP expression; analysis of miR-221 and miR-222 expression was performed by qRT-PCR on GFP+ MC-9 cells. b) Propidium iodide staining and DNA-content analysis of transduced MC-9 cells. c) Quantification of the percentages of transduced MC-9 cells in the different stages of the cell cycle. Each bar is the mean of 4 independent experiments.

Figure 6. SSClo cells are mainly in G1/G0

a) MC-9 cells untransduced (untrans) or transduced with either vector alone (Tween) or miR-expressing vectors were analyzed by FACS for presence of two subsets with different side-scatter (SSChi and SSClo). b) Quantification of the percentages of GFP+ SSChi versus GFP+ SSClo subsets in the transduced cells. Each bar is the mean of 5 different measurements on live, GFP+ cells. c) Propidium iodide staining and DNA content analysis of the sorted SSClo cells.

Figure 7. The effect of miR-221-222 expression on the cell cycle persists under various culture conditions

MC-9 cells transduced with a control vector (Tween) or miR-221-222 (alone or in combination) expressing constructs, were left untreated (first row), or treated with either: i) PMA and ionomycin for 24h (second row); ii) IL-3 withdrawal for 72h (third row); iii) IL-3 withdrawal for 72h followed by re-addition of IL-3 for another 18h (fourth row); iv) 20 ng/ml of SCF for 18h (fifth row). After treatment, cells were stained with propidium iodide and analyzed for DNA content.

Figure 8. Mast cells over-expressing miR-221-222 are impaired in proliferation

a) Thymidine incorporation assay of MC-9 cells untransduced (untr.) or transduced with either the empty Tween vector, or the miR-221, miR-222 and miR-221-222 expressing vectors. Cells were cultured for 4h in a 96 wells-plate with 0.4 μCi 3H-thymidine per well. Cells were then harvested and the amount of incorporated radioactivity determined with a β-counter. The graph shows three independent experiments, each one performed in quadruplicate. b) BrdU incorporation assay of MC-9 cells transduced with either the empty Tween vector, or the miR-221, miR-222 and miR-221-222 expressing vectors. Cells were cultured for 45min in the presence of 10 μM BrdU, before fixation and staining with an anti-BrdU-APC antibody and 7AAD. c) BrdU incorporation assay of MC-9 cells transduced with either the empty Tween vector, or the miR-221, miR-222 and miR-221-222 expressing vectors. Cells were cultured for 48h in the absence of IL-3; IL-3 was then re-added for 14h and 10 μM BrdU was added for the last 30min of culture. Cells were then fixed and stained with an anti-BrdU-APC antibody and 7AAD. Because of the high level of cell death (up to 50%) due to IL-3 withdrawal, dead cells were excluded from the analysis in these particular conditions.

Figure 9. Murine mast cells express two splice variants of p27Kip1 mRNA that differ in the 3′ UTR

a) MC-9 cells were transduced with either vector alone (Tween), or miR-expressing vectors, and were treated with PMA and ionomycin for 24h or left resting. After lysis in Laemmli sample buffer, Western blot analysis was performed for either p27Kip1 or β-tubulin as loading control. b) Schematic representation of two murine p27Kip1 splice variants: the white boxes represent the 5′ and 3′ UTRs; the black boxes the coding sequence and the two grey bars the miR-221-222 binding sites. The positions of the primers used in the RT-PCR in panels c) and d) are also indicated. c) RT-PCR analysis of the expression of the two p27Kip1 splice variants indicated in b). Total RNA was extracted from MC-9 cells untransduced (unt) or transduced with either the empty vector (Tween), or the miR-expressing vectors. After reverse transcription, PCR was performed with the primers indicated in the schematic in b). Actin mRNA was used as control d) Untransduced MC-9 cells were either stimulated with PMA and ionomycin for 36h or left resting; total RNA was extracted and RT-PCR was performed as in c).