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. 2011 May 16:10:55.
doi: 10.1186/1476-4598-10-55.

miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

Nora Bandi  1 , Erik Vassella
Affiliations

miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and Rb-dependent manner

Nora Bandi et al. Mol Cancer. .

Abstract

Background: microRNAs (miRNAs) are small non-coding RNAs that are frequently involved in carcinogenesis. Although many miRNAs form part of integrated networks, little information is available how they interact with each other to control cellular processes. miR-34a and miR-15a/16 are functionally related; they share common targets and control similar processes including G1-S cell cycle progression and apoptosis. The aim of this study was to investigate the combined action of miR-34a and miR-15a/16 in non-small cell lung cancer (NSCLC) cells.

Methods: NSCLC cells were transfected with miR-34a and miR-15a/16 mimics and analysed for cell cycle arrest and apoptosis by flow cytometry. Expression of retinoblastoma and cyclin E1 was manipulated to investigate the role of these proteins in miRNA-induced cell cycle arrest. Expression of miRNA targets was assessed by real-time PCR. To investigate if both miRNAs are co-regulated in NSCLC cells, tumour tissue and matched normal lung tissue from 23 patients were collected by laser capture microdissection and compared for the expression of these miRNAs by real-time PCR.

Results: In the present study, we demonstrate that miR-34a and miR-15a/16 act synergistically to induce cell cycle arrest in a Rb-dependent manner. In contrast, no synergistic effect of these miRNAs was observed for apoptosis. The synergistic action on cell cycle arrest was not due to a more efficient down-regulation of targets common to both miRNAs. However, the synergistic effect was abrogated in cells in which cyclin E1, a target unique to miR-15a/16, was silenced by RNA interference. Thus, the synergistic effect was due to the fact that in concerted action both miRNAs are able to down-regulate more targets involved in cell cycle control than each miRNA alone. Both miRNAs were significantly co-regulated in adenocarcinomas of the lung suggesting a functional link between these miRNAs.

Conclusions: In concerted action miRNAs are able to potentiate their impact on G1-S progression. Thus the combination of miRNAs of the same network rather than individual miRNAs should be considered for assessing a biological response. Since miR-34a and miR-15a/16 are frequently down-regulated in the same tumour tissue, administrating a combination of both miRNAs may also potentiate their therapeutic impact.

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Figures

Figure 1
Figure 1
miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer. (A) miR-16 and miR-34a levels in adenocarcinomas and squamous cell carcinomas relative to matched normal tissues. (B) miR-21 levels in the same tumour samples, relative to matched normal tissue. (C) miR-34a and miR-15a/16 are not able to mutually regulate their expression. H2009 cells were transfected with pre-miR-34a (upper panel) or pre-miR-15a/16 (lower panel) and analysed for the expression of the miRNA counterpart or CDK6 mRNA 42 h post-transfection. Values are relative to the value obtained for the control transfected with precursor control (n = 3). *, P < 0.05.
Figure 2
Figure 2
H2009 cells are refractory to miR-34a-induced cell cycle arrest. (A) DNA content distribution of NSCLC cells transfected with precursor miRNA or precursor control. Cells were treated for 18 h with nocodazole beginning 24 h post-transfection. (B) Percent difference in G1-G0 between cells transfected with pre-miR-34a and cells transfected with precursor control. H2009, A549, n = 3; H1299 and H358, n = 1. (C) mRNA levels of known miR-34a targets. H2009 cells were transfected with pre-miR-34a and harvested 42 h post-transfection (n = 3). Values are relative to the level obtained for the control transfected with precursor control.
Figure 3
Figure 3
miR-34a-induced cell cycle arrest depends on the expression of Rb. (A, D), DNA content distribution by flow cytometry of H2009 (A) and A549 cells (D) treated for 18 h with nocodazole beginning 48 h post-transfection. The percent cells in G1-G0 is shown in the upper panel (n = 3) and a representative histogram of the cell cycle profile is shown in the lower panel. (B, C), Western blot analysis of H2009 (B) and A549 cells (C) subjected to the same conditions as in (A) and (D) using antibodies directed against Rb or phospho-Rb. Protein levels were normalised to α-tubulin.
Figure 4
Figure 4
miR-15a/16 and miR-34a act synergistically to induce cell cycle arrest. (A) Cell cycle analysis of A549 cells transfected with pre-miR-34a and/or pre-miR-15a/16 under non-saturating conditions. Precursors were supplemented with precursor control to yield a total concentration of 2.5 nM per transfection. *, transfection with 2.5 nM precursor control. Cells were treated for 18 h with nocodazole beginning 24 h post-transfection (n = 3). (B) Cell cycle analysis under saturating conditions. A549 cells were transfected with 20 nM precursor or precursor control or co-transfected with 10 nM pre-miR-34a and 10 nM pre-miR-15a/16 and treated for 18 h with nocodazole beginning 24 h (left panel) or 48 h (right panel) post-transfection (n = 3).
Figure 5
Figure 5
No synergism on cell death. (A) Bcl2 expression. A549 or H2009 cells were transfected with 20 nM precursors and harvested 42 h post-transfection. Western blot analysis was performed using a monoclonal antibody against Bcl2. Protein levels were normalized to α-tubulin and presented relative to the level obtained for the control. (B) Time-course of propidium iodide (PI)-positive A549 and H2009 cells by flow cytometry. Cells were transfected with concentrations of precursors as indicated in the legend to Fig. 4B (n = 3). Cells were gated as shown in Additional file 1A. (C) Cleaved caspase-3-positive cells. H2009 cells were analysed for the presence of cleaved caspase 3 by flow cytometry 72 h post-transfection (n = 3). Values are relative to the level obtained for the control transfected with precursor control. As a positive control, cells were treated with UV.
Figure 6
Figure 6
Concerted action of miR-15a/16 and miR-34a on individual mRNA targets. (A) mRNA levels of targets common to both miRNAs. H2009 cells were transfected with pre-miR-15a/16 or pre-miR-34a alone or co-transfected with both pre-miRNAs together (pre-miR-mix) at concentrations as indicated in the figure. (B) Expression level of targets unique to miR-15a/16 or miR-34a. Analysis was performed as described in the legend to Fig. 2C (n = 3). *, p < 0.05.
Figure 7
Figure 7
Synergistic action on cell cycle arrest is due to the down-regulation of unique mRNA targets. A549 (A) or H1299 cells (B) were co-transfected with 20 nM miRNA precursors and 7.8 nM siRNA against CCNE1 and subsequently treated for 18 h with nocodazole beginning 24 h post-transfection. Comparable results were also obtained 48 h post-transfection (data not shown).
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