Limits...
Contact inhibition modulates intracellular levels of miR-223 in a p27kip1-dependent manner.

Armenia J, Fabris L, Lovat F, Berton S, Segatto I, D'Andrea S, Ivan C, Cascione L, Calin GA, Croce CM, Colombatti A, Vecchione A, Belletti B, Baldassarre G - Oncotarget (2014)

Bottom Line: Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells.Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity.Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Oncology 2, CRO, National Cancer Institute, Aviano, Italy.

ABSTRACT
MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.

Show MeSH

Related in: MedlinePlus

p27 binds and stabilizes mature miR-223(A) In vitro miR degradation assay. Expression of miR-223, miR-1, control miR and p27 protein after 0, 90 and 120 minutes of incubation with lysate from p27KO cells is reported. Human recombinant p27 protein was added where indicated. (B) Quantification of miR-223 (left graph) and miR-1 (right graph) levels from the in vitro miR degradation assay described in (A). Data represent the mean of three experiments performed in duplicate and are expressed as fold of miR levels respect to T0. (C) Schematic representation of EGFP-fusion proteins used in RIP assays. (D) Expression of miR-223 and p27 protein using the anti-EGFP or the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. In the lower graph qRT-PCR analyses, evaluating miR-223 binding to p27WT or mutant proteins expressed as fold enrichment respect to miR-223 binding to EGFP transfected cells. (E) Expression of miR-223 and p27 protein using the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. The fold enrichment value (calculated as in D) is reported under the blot. (F) qRT-PCR analyses evaluating miR-223 binding to endogenous p27 protein expressed as fold enrichment respect to miR-223 binding to control IgG in WT and p27KO cells, cultured in exponential growth (EG) or at high confluence (HC). On the right, expression of miR-223 (upper panel) and p27 protein (lower panel) is reported. Different MEF preparations (1 and 2) are indicated. p values were obtained by unpaired student t-test, using either Excel or Prism software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4012735&req=5

Figure 5: p27 binds and stabilizes mature miR-223(A) In vitro miR degradation assay. Expression of miR-223, miR-1, control miR and p27 protein after 0, 90 and 120 minutes of incubation with lysate from p27KO cells is reported. Human recombinant p27 protein was added where indicated. (B) Quantification of miR-223 (left graph) and miR-1 (right graph) levels from the in vitro miR degradation assay described in (A). Data represent the mean of three experiments performed in duplicate and are expressed as fold of miR levels respect to T0. (C) Schematic representation of EGFP-fusion proteins used in RIP assays. (D) Expression of miR-223 and p27 protein using the anti-EGFP or the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. In the lower graph qRT-PCR analyses, evaluating miR-223 binding to p27WT or mutant proteins expressed as fold enrichment respect to miR-223 binding to EGFP transfected cells. (E) Expression of miR-223 and p27 protein using the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. The fold enrichment value (calculated as in D) is reported under the blot. (F) qRT-PCR analyses evaluating miR-223 binding to endogenous p27 protein expressed as fold enrichment respect to miR-223 binding to control IgG in WT and p27KO cells, cultured in exponential growth (EG) or at high confluence (HC). On the right, expression of miR-223 (upper panel) and p27 protein (lower panel) is reported. Different MEF preparations (1 and 2) are indicated. p values were obtained by unpaired student t-test, using either Excel or Prism software.

Mentions: These data prompted us to directly test whether p27 affected miR-223 stability. To this aim, we used an in vitro degradation assay in which cell lysates from p27 cells were used to test the half-life of a synthetic miR-223 oligo, in the presence or not of recombinant p27. This assay demonstrated that recombinant p27 protein was able to significantly lengthen the half-life of miR-223, but not of control-miR or of the un-related miR-1 (Figure 5A and B), suggesting that p27 directly bound and protected from degradation miR-223. This possibility was supported by in silico analyses (BindN prediction program), showing that both human and mouse p27 proteins contains several putative RNA-binding domains (Figure 5C and Supplementary Figure 3). Using RNA-immunoprecipitation (RIP) assay, we could assess that human p27 was able to bind miR-223 in a cytoplasmic and CDK-independent manner, by a region comprised between aminoacids (aa) 86 and 154 (Figure 5D and Supplementary Figure 3C-E). Two putative RNA-binding sites are present in this region and one of them is highly conserved among mammals (Supplementary Figure 3F). The most conserved RNA-binding site was the region comprised between aa 90 and 103 (Figure 5D and Supplementary Figure 3F). In silico analyses demonstrated that three point mutations in R93, P95 and K96 would be sufficient to completely disrupt this binding site (Supplementary Figure 3G). We thus generated this mutant (hereafter called p27MUT1) and demonstrated that it barely binds miR-223, with an affinity below the threshold we set as significant when the fold enrichment was calculated (Figure 5E). Next, we tested the possibility that also the region between aa 134 and 142 could bind miR-223. In doing so, we took advantage of the notion that in human breast cancer it has been recently isolated a mutant form of p27 carrying one base deletion at codon 134, resulting in the frameshift of p27 open reading frame and in a truncated protein [28]. We generated this mutant, named p27K134fs and our RIP assay demonstrated that it is still able to bind miR-223, although with very low affinity (Figure 5E). When the two RNA-binding site where concomitantly destroyed (p27dMUT mutant) (Figure 5E and Supplementary Figure 3F) we observed no binding between p27 and miR-223. These data suggested that miR-223 could primarily bind the region between aa 90 and 103 that it is highly conserved among species, and that the region between aa 134 and 142 could participate in the binding (Supplementary Figure 3F). Then, a RIP performed on endogenous p27 immunoprecipitated from HC or EG WT and p27KO MEFs proved the physiological significance of this interaction showing that miR-223 was readily recovered only in WT HC cells (Figure 5F).


Contact inhibition modulates intracellular levels of miR-223 in a p27kip1-dependent manner.

Armenia J, Fabris L, Lovat F, Berton S, Segatto I, D'Andrea S, Ivan C, Cascione L, Calin GA, Croce CM, Colombatti A, Vecchione A, Belletti B, Baldassarre G - Oncotarget (2014)

p27 binds and stabilizes mature miR-223(A) In vitro miR degradation assay. Expression of miR-223, miR-1, control miR and p27 protein after 0, 90 and 120 minutes of incubation with lysate from p27KO cells is reported. Human recombinant p27 protein was added where indicated. (B) Quantification of miR-223 (left graph) and miR-1 (right graph) levels from the in vitro miR degradation assay described in (A). Data represent the mean of three experiments performed in duplicate and are expressed as fold of miR levels respect to T0. (C) Schematic representation of EGFP-fusion proteins used in RIP assays. (D) Expression of miR-223 and p27 protein using the anti-EGFP or the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. In the lower graph qRT-PCR analyses, evaluating miR-223 binding to p27WT or mutant proteins expressed as fold enrichment respect to miR-223 binding to EGFP transfected cells. (E) Expression of miR-223 and p27 protein using the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. The fold enrichment value (calculated as in D) is reported under the blot. (F) qRT-PCR analyses evaluating miR-223 binding to endogenous p27 protein expressed as fold enrichment respect to miR-223 binding to control IgG in WT and p27KO cells, cultured in exponential growth (EG) or at high confluence (HC). On the right, expression of miR-223 (upper panel) and p27 protein (lower panel) is reported. Different MEF preparations (1 and 2) are indicated. p values were obtained by unpaired student t-test, using either Excel or Prism software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4012735&req=5

Figure 5: p27 binds and stabilizes mature miR-223(A) In vitro miR degradation assay. Expression of miR-223, miR-1, control miR and p27 protein after 0, 90 and 120 minutes of incubation with lysate from p27KO cells is reported. Human recombinant p27 protein was added where indicated. (B) Quantification of miR-223 (left graph) and miR-1 (right graph) levels from the in vitro miR degradation assay described in (A). Data represent the mean of three experiments performed in duplicate and are expressed as fold of miR levels respect to T0. (C) Schematic representation of EGFP-fusion proteins used in RIP assays. (D) Expression of miR-223 and p27 protein using the anti-EGFP or the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. In the lower graph qRT-PCR analyses, evaluating miR-223 binding to p27WT or mutant proteins expressed as fold enrichment respect to miR-223 binding to EGFP transfected cells. (E) Expression of miR-223 and p27 protein using the anti-p27 antibodies (Ab#Cl57), in lysates and RIP from MDA-MB-231-miR-223 cells transfected with the indicated p27 constructs and immunoprecipitated using an anti-EGFP or control IgG antibodies, as indicated. The fold enrichment value (calculated as in D) is reported under the blot. (F) qRT-PCR analyses evaluating miR-223 binding to endogenous p27 protein expressed as fold enrichment respect to miR-223 binding to control IgG in WT and p27KO cells, cultured in exponential growth (EG) or at high confluence (HC). On the right, expression of miR-223 (upper panel) and p27 protein (lower panel) is reported. Different MEF preparations (1 and 2) are indicated. p values were obtained by unpaired student t-test, using either Excel or Prism software.
Mentions: These data prompted us to directly test whether p27 affected miR-223 stability. To this aim, we used an in vitro degradation assay in which cell lysates from p27 cells were used to test the half-life of a synthetic miR-223 oligo, in the presence or not of recombinant p27. This assay demonstrated that recombinant p27 protein was able to significantly lengthen the half-life of miR-223, but not of control-miR or of the un-related miR-1 (Figure 5A and B), suggesting that p27 directly bound and protected from degradation miR-223. This possibility was supported by in silico analyses (BindN prediction program), showing that both human and mouse p27 proteins contains several putative RNA-binding domains (Figure 5C and Supplementary Figure 3). Using RNA-immunoprecipitation (RIP) assay, we could assess that human p27 was able to bind miR-223 in a cytoplasmic and CDK-independent manner, by a region comprised between aminoacids (aa) 86 and 154 (Figure 5D and Supplementary Figure 3C-E). Two putative RNA-binding sites are present in this region and one of them is highly conserved among mammals (Supplementary Figure 3F). The most conserved RNA-binding site was the region comprised between aa 90 and 103 (Figure 5D and Supplementary Figure 3F). In silico analyses demonstrated that three point mutations in R93, P95 and K96 would be sufficient to completely disrupt this binding site (Supplementary Figure 3G). We thus generated this mutant (hereafter called p27MUT1) and demonstrated that it barely binds miR-223, with an affinity below the threshold we set as significant when the fold enrichment was calculated (Figure 5E). Next, we tested the possibility that also the region between aa 134 and 142 could bind miR-223. In doing so, we took advantage of the notion that in human breast cancer it has been recently isolated a mutant form of p27 carrying one base deletion at codon 134, resulting in the frameshift of p27 open reading frame and in a truncated protein [28]. We generated this mutant, named p27K134fs and our RIP assay demonstrated that it is still able to bind miR-223, although with very low affinity (Figure 5E). When the two RNA-binding site where concomitantly destroyed (p27dMUT mutant) (Figure 5E and Supplementary Figure 3F) we observed no binding between p27 and miR-223. These data suggested that miR-223 could primarily bind the region between aa 90 and 103 that it is highly conserved among species, and that the region between aa 134 and 142 could participate in the binding (Supplementary Figure 3F). Then, a RIP performed on endogenous p27 immunoprecipitated from HC or EG WT and p27KO MEFs proved the physiological significance of this interaction showing that miR-223 was readily recovered only in WT HC cells (Figure 5F).

Bottom Line: Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells.Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity.Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Oncology 2, CRO, National Cancer Institute, Aviano, Italy.

ABSTRACT
MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.

Show MeSH
Related in: MedlinePlus