microRNAome changes in bystander three-dimensional human tissue models suggest priming of apoptotic pathways

Abstract

The radiation-induced bystander effect (RIBE) is a phenomenon whereby unexposed cells exhibit molecular symptoms of stress exposure when adjacent or nearby cells are traversed by ionizing radiation (IR). Recent data suggest that RIBE may be epigenetically mediated by microRNAs (miRNAs), which are small regulatory molecules that target messenger RNA transcripts for translational inhibition. Here, we analyzed microRNAome changes in bystander tissues after α-particle microbeam irradiation of three-dimensional artificial human tissues using miRNA microarrays. Our results indicate that IR leads to a deregulation of miRNA expression in bystander tissues. We report that major bystander end points, including apoptosis, cell cycle deregulation and DNA hypomethylation, may be mediated by altered expression of miRNAs. Specifically, c-MYC-mediated upregulation of the miR-17 family was associated with decreased levels of E2F1 and RB1, suggesting a switch to a proliferative state in bystander tissues, while priming these cells for impending death signals. Upregulation of the miR-29 family resulted in decreased levels of its targets DNMT3a and MCL1, consequently affecting DNA methylation and apoptosis. Altered expression of miR-16 led to changes in expression of BCL2, suggesting modulation of apoptosis. Thus, our data clearly show that miRNAs play a profound role in the manifestation of late RIBE end points. In summary, this study creates a roadmap for understanding the role of microRNAome in RIBE and for developing novel RIBE biomarkers.

Fig. 1.

Tissue dimensions and irradiation setup. A narrow α-particle beam irradiated the EpiAirway tissue through the membrane to deliver approximately five α-particles to the nucleus of each cell in a single plane across the tissue base. Bystander tissues were harvested on either side of the irradiated plane for the microarray experiments.

Fig. 2.

Heat map of ANOVA (P < 0.05) expression analysis for all treatments. Vertical columns denote days after irradiation for the mock treatment and the bystander (BS) tissues. Control (CT)-unirradiated sample. The white box includes co-regulated members of the miR-17-92 family. Red boxes highlight important miRNAs discussed in the article. Each line represents an independent biological replicate.

Fig. 3.

Upregulation of cMYC levels in bystander tissues. Samples were loaded in a random manner to ensure lack of bias. For presentation purposes, after scanning pictures were arranged in the following order: mock 8h, bystander 8h, mock 3d, bystander 3d and bystander 5d. BS—bystander, 8h—8 h after irradiation, 3d—3 days after irradiation and 5d—5 days after irradiation.

Fig. 4.

Altered expression of miRNA targets in bystander cells. (A) Targets of miR-106. (B) Targets of miR-16. (C) Targets of miR-29. Samples were loaded in a random manner to ensure lack of bias. For presentation purposes, after scanning pictures were arranged in the following order: mock 8h, bystander 8h, mock 3d, bystander 3d and bystander 5d. BS, bystander, 8h—8 h after irradiation, 3d—3 days after irradiation and 5d—5 days after irradiation.

Fig. 5.

Increase of apoptotic cells in bystander tissues following microbeam irradiation. IR-induced apoptotic cell death was scored in side areas of the sections containing unirradiated bystander cells; *significantly different from the control, P < 0.05, Student’s t-test. Cells positive for cleaved caspase-3 were recorded at various times post-IR in Air-100 epithelium. Representative images of apoptotic cells in Air-100 bystander epithelium positive for both cleaved caspase-3 (red) and γ-H2AX (green), 10 μm frozen sections, 40×, oil immersion. Cleaved caspase-3 is stained with Alexa 546 (red) and γ-H2AX is stained with Alexa 488 (green).

Fig. 6.

miRNAs that are involved in regulation of important bystander effect end points by targeting crucial regulator proteins.