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. 2011 Jun;18(6):974-84.
doi: 10.1038/cdd.2010.164. Epub 2011 Jan 14.

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival

E A Afanasyeva  1 , P Mestdagh, C Kumps, J Vandesompele, V Ehemann, J Theissen, M Fischer, M Zapatka, B Brors, L Savelyeva, V Sagulenko, F Speleman, M Schwab, F Westermann
Affiliations

MicroRNA miR-885-5p targets CDK2 and MCM5, activates p53 and inhibits proliferation and survival

E A Afanasyeva et al. Cell Death Differ. 2011 Jun.

Abstract

Several microRNA (miRNA) loci are found within genomic regions frequently deleted in primary neuroblastoma, including miR-885-5p at 3p25.3. In this study, we demonstrate that miR-885-5p is downregulated on loss of 3p25.3 region in neuroblastoma. Experimentally enforced miR-885-5p expression in neuroblastoma cell lines inhibits proliferation triggering cell cycle arrest, senescence and/or apoptosis. miR-885-5p leads to the accumulation of p53 protein and activates the p53 pathway, resulting in upregulation of p53 targets. Enforced miR-885-5p expression consistently leads to downregulation of cyclin-dependent kinase (CDK2) and mini-chromosome maintenance protein (MCM5). Both genes are targeted by miR-885-5p via predicted binding sites within the 3'-untranslated regions (UTRs) of CDK2 and MCM5. Transcript profiling after miR-885-5p introduction in neuroblastoma cells reveals alterations in expression of multiple genes, including several p53 target genes and a number of factors involved in p53 pathway activity. Taken together, these data provide evidence that miR-885-5p has a tumor suppressive role in neuroblastoma interfering with cell cycle progression and cell survival.

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Figures

Figure 1
Figure 1
The miR-885-5p gene at 3p25.3 is expressed at low level in primary neuroblastomas with segmental 3p loss. (a) Patient characteristics are shown for the 193 primary neuroblastomas analyzed on either 44 or 105 K oligo microarrays. The percentage of tumors with heterozygous 3p25.3/miR-885-5p deletion is indicated in parenthesis. (b) Expression of miR transcripts was verified using RT-qPCR in 60 primary neuroblastomas, and is shown as box plots. MNA–MYCN-amplified neuroblastomas. ΔCq values were multiplied by −1 to represent relative expression of miR-885-5p and miR-331-3p. miR-885-5p: median ΔCq × −1=1.83, range −0.04–6.43; miR-331-3p: median ΔCq × −1=−7.31, range −8.57 to −4.35). (c) Neuroblastoma cell lines used in the screen. The MYCN amplification, TP53/INK4a/ARF and 3p status are listed as reported previously13, 14 or assessed in this study; An asterisk (*) denotes – age of the patient – 10 years, stage – 4. (d) Expression of miR-885-5p and miR-331-3p was assessed in nine established neuroblastoma cell lines using RT-qPCR (bar graphs). miR-885-5p ΔCq × −1 median −8.1, range −6.3 to −12.4; miR-331-3p ΔCq × −1 median −4.3, range −3 to −5.2
Figure 2
Figure 2
miR-885-5p inhibits neuroblastoma proliferation and survival. (a) TP53 wild-type IMR32, SH-EP, KELLY and TP53 mutant HDN33, SK-N-BE(2)c cells were transfected with miR-885-5p, miR-331-3p mimics, control miRNA (each 30 n), Lipofectamine 2000 (vehicle) or left untreated. Cell viability was measured by Alamar Blue assay. The data are represented as the means±S.D. (a; insert) SA-ß-Gal assays were performed 4 days after transfection; the positively stained cells were counted (mean percentages±S.D. of three experiments are presented). (b) IMR32, SH-EP, KELLY and SK-N-BE(2)c were transfected with miRNA mimics and assayed for anchorage-independent growth in soft agar after 2 weeks in culture. Colonies were fixed and stained with crystal violet and counted using ImageJ software. The graphs (right) show mean percentages of colonies number±S.D. of three experiments. (c) Cell cycle profiles of controls and cells transfected with miRNA mimics were determined using FACS analysis. (d) The sub-G1 fraction of neuroblastoma cell lines transfected with miRNA mimics or control miRNA transfectants were detected at 4-days post transfection using FACS analysis by detection of cells with a hypodiploid DNA content (bar=mean percentage of sub-G1±S.D.). Phosphatidylserine externalization was analyzed by annexin V/PI staining, the results show the percentage of annexin V-positive/PI negative and annexin V/PI double positive cells
Figure 3
Figure 3
miR-885-5p induces p53 and downregulates CDK2 and MCM5. (a and b) Protein expression of several cell cycle and apoptosis regulators, including potential miR-885-5p targets, is shown in western blots of neuroblastoma cells lines at 4 days after transfection of miR-885-5p, miR-331-3p or controls. MCM3 cleavage products are indicated (*). Densitometry analysis was performed using ImageJ software; the relative densitometry values are shown on top
Figure 4
Figure 4
miR-885-5p directly targets CDK2 and MCM5. Predicted miR-885-5p target sites in the CDK2 and MCM5 3′-UTRs. Lines indicate perfect matches; colons, G:U pairs. Luciferase reporter assays of stably transfected cells and miRNA-mimic-transfected cells (indicated on x-axis) transiently transfected with either empty vector or vector containing wild-type or mutant miR-885-5p binding sites (indicated as bar color). Firefly luciferase values were normalized to renilla activity. The highest normalized luciferase activity of the stable transfected cells or cells transfected with miRNA mimics was set to 1, and values are reported as fold increase (mean relative luciferase activity±S.D.). *P=<0.1; **P=<0.05; ***P=<0.01 versus vector with mutant miR-885-5p binding sites
Figure 5
Figure 5
CDK2 and MCM5 knockdown mimic miR-885-5p-induced phenotype. (a) TP53 wild-type IMR32, SH-EP, KELLY and TP53 mutant HDN33, SK-N-BE(2)c cells were transfected with anti CDK2 (5 n), anti MCM5 (5 n), anti CDK2 (2.5 n)+ anti MCM5 (2.5 n) siRNAs or control siRNA (5 n) with Lipofectamine 2000. Cell viability was measured at 24, 48 and 72 h after transfection by Alamar Blue assay. The data are represented as the mean±S.D. (b) siRNA transfected cells were assayed for anchorage-independent growth in soft agar after 2 weeks in culture. Colonies were counted using ImageJ software (mean percentages of colonies number±S.D. of three experiments are presented in the graphs (right)). (c) SA-ß-Gal assays were performed at 4 days after transfection; the positively stained cells were counted (mean percentages±S.D. of three experiments are presented). Cell cycle profiles of controls and cells transfected with siRNAs were determined using FACS analysis. The sub-G1 fraction of neuroblastoma cell lines transfected with miRNA mimics or control miRNA transfectants were detected at 4-days post transfection by FACS analysis by detection of cells with a hypodiploid DNA content (bar=mean percentage of sub-G1±S.D.). Phosphatidylserine externalization was analyzed by annexin V/PI staining, the percentage of annexin V-positive and annexin V/PI double-positive cells are indicated. (d) protein expression of MCM5, CDK2, p21waf1, p53, PUMA and E2F1 from siRNA-transfected cells is shown in western blots. The relative densitometry values are shown
Figure 6
Figure 6
miR-885-5p-induced growth arrest is p53 dependent in TP53 wild-type neuroblastoma cells. (a) IMR32, SH-EP and KELLY cells were transfected with siRNA against p53 or control siRNA using Lipofectamine 2000 and transfected with miRNA mimics after 24 h. p53 knockdown efficiency was evaluated at day 4 after transfection (insert below). Cell viability was measured by Alamar Blue assay for 24, 48 and 72 h after miRNA mimic transfection (upper panel). The data are represented as the means±S.D. of three experiments. The cell cycle profiles were performed by FACS analysis (middle panel). (a, insert) p53 knockdown efficiency is shown in western blots in IMR32, KELLY and SH-EP. (b) p53 knockdown and control siRNA transfected IMR32, KELLY and SH-EP cells were transfected with miRNA mimics and assayed for anchorage-independent growth in soft agar after 2 weeks in culture. Colonies were fixed and stained with crystal violet and counted using ImageJ software. The graphs show mean percentages of colonies number±S.D. of three experiments. (c) Protein expression of p21waf1, PUMA and p53 is shown in western blots of p53 knockdown and control siRNA transfected IMR32, KELLY and SH-EP cells at 3 days after transfection of miRNA mimics. (d) IMR32 and SK-N-BE(2)c were transfected with antimiR-885-5p or control antimiR (30 n) and assayed for anchorage-independent growth for 2 weeks. Colonies were counted using ImageJ software (mean percentages of colonies number±S.D. of three experiments are presented). Protein expression of p53, p21waf1, PUMA, CDK2 and MCM5 in antimiR-transfected IMR32 and SK-N-BE(2)c cells is shown in western blots. The relative densitometry values are shown on top of the western blot panels
Figure 7
Figure 7
A model for miR-885-5p function. Proposed model for miR-885-5p function via CDK2 and MCM5 downregulation in neuroblastoma. miR-885-5p downregulates CDK2 leading to p53 activation in neuroblastoma cells with wild-type TP53. miR-885-5p-triggered CDK2 and MCM5 downregulation reduces cell renewal capacity and favors apoptosis and senescence in neuroblastoma with either wild-type or non-functional TP53
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