Oncogenic microRNA 17-92 cluster is regulated by epithelial cell adhesion molecule and could be a potential therapeutic target in retinoblastoma

Abstract

Purpose: Several miRNAs have been reported as candidate oncogenes and tumor suppressors, which are involved in the pathways specifically altered during tumorigenesis or metastasis. The miR 17-92 cluster located in 13q31 locus might contribute to retinoblastoma (RB) oncogenesis as 13q31 is amplified often in RB. We attempted to identify the factors involved in the regulation of miR 17-92 cluster in RB.

Methods: Real-time quantitative reverse transcriptase PCR was performed to study the expression of the miR 17-92 cluster in primary RB tumors and in Y79 cells after epithelial cell adhesion molecule (EpCAM) silencing. EpCAM was silenced using siRNA and confirmed by western blotting. The Y79 cells were transfected with individual and mixed antagomirs and studied the cell viability by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, invasion by matrigel analysis and caspase-3 expression by flow cytometry.

Results: The relative expression of miR 17-92 cluster, compared to that of a normal retina, ranged from 25 to 220 fold (p<0.0001), miR-18 being highly expressed in RB. Post EpCAM silencing resulted in a significant decrease (p<0.01) in the expression of the miR 17-92 cluster by 4 to eightfold in Y79 cells. Y79 cells transfected with an antagomirs mix (all 5 miRNAs) showed decreased cell viability (p<0.001) and cell invasion (p<0.001). Similarly, Y79 cells treated with antagomirs mix showed increased expression of caspase-3 (p<0.001), which confirms the anti-proliferative effect of antagomirs.

Conclusions: This study has showed varied expression of the miR17-92 cluster in primary RB tumors. EpCAM influences miR 17-92 cluster expression in retinoblastoma. In addition, we showed that the miR 17-92 cluster plays a role in RB cell proliferation and invasion. Therefore, targeting the miRNA 17-92 cluster may be beneficial for controlling Y79/RB cell proliferation and invasion.

Figure 1

A graph showing the expression of miR 17–92 cluster in primary retinoblastoma (RB) tumors. The blue bars represent RB tumors and the red bars represent Y79 cells. The error bars represent the varying expression of microRNA in 19 tumors.

Figure 2

Correlation between miR-18a and EpCAM expression in RB tumors. A: A bar diagram showing the expression of EpCAM in retinoblastoma (RB) tumor samples. The expression ranged from 12-fold to 200 fold compared to normal control retina samples. B: The plot showing the correlation between EpCAM and miR-18a in primary RB tumors. The correlation R value is equal to 0.81 (strong correlation).

Figure 3

siRNA mediated EpCAM downregulation and its effect on miR 17–92 expression. A: The western blot shows the down-regulation of EpCAM in Y79 cells treated with EpCAM specific siRNA. The lower panel shows the Beta actin expression as loading controls. B: QPCR shows the change in the expression of the miR 17–92 cluster in Y79 cells treated with EpCAM siRNA. There is a significant down-regulation of the miR 17–92 cluster by 4 to eightfold (*p<0.01).

Figure 4

Effect of antagomirs on miR 17–92 cluster, cell viability and cellular invasion. A: The bar diagram shows the expression of miR 17–92 cluster in Y79 cells treated with specific antagomirs. There is a significant down-regulation of miR 17–92 clusters when treated with antagomirs. B: MTT assay graph showing the Y79 cell viability after 48 h of treatment with individual and mix antagomirs. There is a significant decrease in cell viability when treated with antagomirs-17 (*p<0.01), antagomirs-19b (*p<0.01) and antagomirs mix (*p<0.001). C: shows the effect of antagomirs (17–92) on Y79 cellular invasion process as analyzed by Matrigel invasion assay. There is a significant decrease in cell invasion when treated with antagomirs-19b (*p<0.05) and antagomirs mix (**p<0.001)

Figure 5

Transfection of antagomir mix induces caspase-3 expression in RB cells. A: Shows the flow cytometry density plot for caspase-3 expression in Y79 cells treated with control antagomir. The percentage of caspase-3 is negligible (less than 1%). B: Shows the flow cytometry density plot for caspase-3 expression in Y79 cells treated with antagomirs mix. There is a significant increase in caspase-3 expression (32%) in Y79 cells treated with antagomirs mix (*p<0.001).