MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

Abstract

Background: Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.

Results: We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.

Conclusions: MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

Figure 1

Growth curves for Kelly (A) and SK-N-AS (B) following transfection with the anti-miR-184 (×3 biological replicates). A scrambled anti-miR was used as negative control.

Figure 2

(A) Predicted alignment of miR-184 to the mRNA 3'UTR region of AKT2, as predicted by the Sanger miR Registry. (B) AKT2 mRNA levels were significantly different (p < 0.002) between tumours with high miR-184 levels (predominantly MNA tumors)(n = 10) and tumours with low miR-184 levels (predominantly non-MNA)(n = 9). AKT2 mRNA levels were also significantly different (p < 0.035) between tumours with MNA (n = 10) versus non-MNA (n = 9).

Figure 3

(A) RT-qPCR analysis of miR-184 in four neuroblastoma cell lines. RNU66 was used as an endogenous control and expression levels are relative to SH-SY5Y, set as 1.0. AKT2 mRNA (B) and protein (C) levels in the same four neuroblastoma cell lines (SK-N-AS, SK-N-BE, Kelly, SH-SY5Y). Actin was used as an endogenous loading control for the western blot, while RPLPO was used as endogenous control for RT-qPCR, all values are relative to SK-N-AS. Note that AKT2 mRNA levels show an inverse relationship to the endogenous miR-184 levels.

Figure 4

Relative AKT2 mRNA levels following transfection of miR-184 mimics (pre-miR-184), anti-miR-184, or a scrambled oligo (NC) into (A) Kelly and (B) SK-N-AS cells at 24, 48 and 72 hrs after transfection. RT-qPCR results were normalised to RPLPO ribosomal protein and are relative to the negative control at 24 hrs. Western Blot (C) representing protein levels of AKT2 in Kelly cells 72 hrs after transfection with the miR-184 mimics, a negative control oligo, or anti-miR-184. β-Actin was used for the endogenous control. (D) pMir-Reporter containing the AKT2 binding site for miR-184 in the 3' UTR region of the luciferase gene co-transfected into Kelly cells with a scrambled negative control oligonucleotide (left) or mature miR-184 mimic (middle). The right bar shows the result of co-transfection with the pMir-Reporter containing the mutated AKT2 binding site for miR-184 co-transfected into Kelly cells with mature miR-184 mimic. Luciferase values were normalized to B-galactosidase on a second plasmid that was co-transfected into each cell line and all values are relative to the negative control (left).

Figure 5

(A) Growth curves following transfection with each AKT2 siRNA and negative control for (A) Kelly (p values for each siRNA ranged from 0.01 to 0.03) and (B) SK-N-AS (p values for each siRNA ranged from 0.007 to 0.03). Growth curves (C) for Kelly cells following transfection with various combinations of AKT2 expression vector, empty vector, miR-184 mimics or negative control (NC) oligonucleotides. The highest number of cells was produced by either the AKT2 expression vector alone or in combination with a negative control oligonucleotide. These cells had both endogenous and ectopic AKT2 (Additional File 5). Co-transfection with the AKT2 expression plasmid (lacking the miR-184 target site) and miR-184 mimics yielded a cell number curve that was not statistically different from co-transfection with a negative control oligonucleotide and the empty expression vector. The former cell population had ectopic but not endogenous AKT2 activity, while the latter cell population had endogenous, but no ectopic AKT2. Both of these cell number curves were significantly different from the other curves (p < 0.003). Transfection of miR-184 mimics by themselves or in combination with the empty vector significantly impacted cell growth, as these cells had no ectopic AKT2 and endogenous AKT2 was inhibited.

Figure 6

FACs analysis of annexin V staining in Kelly cells. (A) transfection with negative control oligonucleotide and (B) siRNA mediated knock down of AKT2 using siRNA. Cells were harvested for detection of externalised annexin V (apoptotic cells) and PI staining of DNA (necrotic cells). Double-stained cells (upper right quadrant) indicate cells in late apoptosis. The resultant increase in apoptosis caused by siRNA inhibition of AKT2 was highly significant (P < 0.0001). Analysis of caspase 3/7 activity in Kelly (C) and SK-N-AS (D) cells following siRNA knockdown of AKT2 relative to negative control (NC).