MicroRNA expression after ionizing radiation in human endothelial cells

Abstract

Background: Endothelial cells (EC) in tumor and normal tissue constitute critical radiotherapy targets. MicroRNAs have emerged as master switchers of the cellular transcriptome. Here, we seek to investigate the role of miRNAs in primary human dermal microvascular endothelial cells (HDMEC) after ionizing radiation.

Methods: The microRNA status in HDMEC after 2 Gy radiation treatment was measured using oligo-microarrays covering 361 miRNAs. To functionally analyze the role of radiation-induced differentially regulated miRNAs, cells were transfected with miRNA precursor or inhibitor constructs. Clonogenic survival and proliferation assays were performed.

Results: Radiation up-regulated miRNA expression levels included let-7g, miR-16, miR-20a, miR-21 and miR-29c, while miR-18a, miR-125a, miR-127, miR-148b, miR-189 and miR-503 were down-regulated. We found that overexpression or inhibition of let-7g, miR-189, and miR-20a markedly influenced clonogenic survival and cell proliferation per se. Notably, the radiosensitivity of HDMEC was significantly influenced by differential expression of miR-125a, -127, -189, and let-7g. While miR-125a and miR-189 had a radioprotective effect, miR-127 and let-7g enhanced radiosensitivity in human endothelial cells.

Conclusion: Our data show that ionizing radiation changes microRNA levels in human endothelial cells and, moreover, exerts biological effects on cell growth and clonogenicity as validated in functional assays. The data also suggest that the miRNAs which are differentially expressed after radiation modulate the intrinsic radiosensitivity of endothelial cells in subsequent irradiations. This indicates that miRNAs are part of the innate response mechanism of the endothelium to radiation.

Figure 1

Differentially expressed miRNAs in HDMEC. The figure includes those miRNAs which are listed in Tab. 1. The heat-map was generated via a t-test of microarray data of radiation treated versus untreated cells. Each column represents the medium value of three biological replicates. Colours represent log2 values from -1.5 to 1.5.

Figure 2

Clonogenic survival of HDMEC after transfection with miR-189 precursor or inhibitor. Cells were treated as described in 'Methods'. Bar charts: Left sided: Influence of the transfected molecules on clonogenic survival without RT. Negative controls were set 100%. Right sided: Survival of irradiated cells (2 Gy) after transfection with the miRNA precursor (pre-miR) or inhibitor (anti-miR). Only miR transfected samples without irradiation were set 100%. Bars: Mean (n = 3) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.

Figure 3

Clonogenic survival of HDMEC after transfection with let-7g precursor or inhibitor. Cells were treated as described in 'Methods'. Bar charts: Left sided: Influence of the transfected molecules on clonogenic survival without RT. Negative controls were set 100%. Right sided: Survival of irradiated cells (2 Gy) after transfection with the miRNA precursor (pre-miR) or inhibitor (anti-miR). Only miR transfected samples without irradiation were set 100%. Bars: Mean (n = 3) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.

Figure 4

Clonogenic survival of HDMEC after transfection with miR-20a precursor or inhibitor. Cells were treated as described in 'Methods'. Bar charts: Left sided: Influence of the transfected molecules on clonogenic survival without RT. Negative controls were set 100%. Right sided: Survival of irradiated cells (2 Gy) after transfection with the miRNA precursor (pre-miR) or inhibitor (anti-miR). Only miR transfected samples without irradiation were set 100%, except for pre-miR-20a. Bars: Mean (n = 3) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.

Figure 5

Clonogenic survival of HDMEC after transfection with miR-125a precursor or inhibitor. Cells were treated as described in 'Methods'. Bar charts: Left sided: Influence of the transfected molecules on clonogenic survival without RT. Negative controls were set 100%. Right sided: Survival of irradiated cells (2 Gy) after transfection with the miRNA precursor (pre-miR) or inhibitor (anti-miR). Only miR transfected samples without irradiation were set 100%. Bars: Mean (n = 3) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.

Figure 6

Clonogenic survival of HDMEC after transfection with miR-127 precursor or inhibitor. Cells were treated as described in 'Methods'. Bar charts: Left sided: Influence of the transfected molecules on clonogenic survival without RT. Negative controls were set 100%. Right sided: Survival of irradiated cells (2 Gy) after transfection with the miRNA precursor (pre-miR) or inhibitor (anti-miR). Only miR transfected samples without irradiation were set 100%. Bars: Mean (n = 3) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.

Figure 7

Clonogenic survival of HDMEC after transfection with miR-148b precursor or inhibitor. Cells were treated as described in 'Methods'. Bar charts: Left sided: Influence of the transfected molecules on clonogenic survival without RT. Negative controls were set 100%. Right sided: Survival of irradiated cells (2 Gy) after transfection with the miRNA precursor (pre-miR) or inhibitor (anti-miR). Only miR transfected samples without irradiation were set 100%. Bars: Mean (n = 3) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.

Figure 8

Proliferation assay of irradiated cells. HDMEC were treated as described in 'Methods'. Panels A-C show the proliferation data of cells pre-treated with precursor or inhibitor molecules of miR-189, let-7g and miR-20a. The bar charts present the mean proliferation of HDMEC after irradiation, dependent on the pre-treatment. Fluorescence values are set in percentage related to non-irradiated cells (100%). Bars: Mean (n = 24) with SD. *: P < 0.05 versus the pre-miR or anti-miR negative control.