In this study, we analyzed microRNAome changes in bystander tissues in an ‘in vivo
’ EpiAirway human tissue model. Bystander tissues in this model display profound cellular and molecular changes (9
). Specifically, previous research showed an increase in apoptosis, DNA DSBs, micronucleus formation and cellular senescence as well as a decrease in global genome methylation. Additionally, RIBE is associated with deregulation of cell cycle control. Given that miRNAs are known to regulate some of these processes, we investigated the roles that miRNAs may play in the bystander response. Importantly, we found that major RIBE end points—apoptosis, cell cycle deregulation and global genome hypomethylation may be mediated by altered expression of miRNAs ().
miRNAs that are involved in regulation of important bystander effect end points by targeting crucial regulator proteins.
Furthermore, we also show that altered miRNA expression in bystander tissues may be due to the bystander-induced expression of c-MYC. c-MYC is a widely studied gene involved in the control of cell size, cell cycle, proliferation and apoptosis (43
). It has been shown that the proto-oncogene c-MYC transcription factor binds the miR-17-92 cluster’s promoter region, inducing these miRNAs to target E2F transcripts (35
). The current paradigm is that the miR-17-92 cluster works as a regulator of a positive feedback loop between c-MYC and E2F transcription factors, preventing a reciprocal positive feedback from ‘runaway’ regulation (44
). The upregulation of the miR-17-92 cluster via c-MYC to cause elevated expression levels of miR-17 and -20a is thought to indicate environmental signals to switch the cell to a proliferative state (44
). Our study shows that upregulation of MYC and the miR-17-92 cluster and downregulation of E2F1 in bystander tissues, support the current paradigm. Furthermore, we found that the upregulation of miR-106 was correlated with downregulation of RB1.
It is interesting to speculate on the function of c-MYC activity in bystander tissues. Increased levels of c-MYC in the cell are most often associated with deregulated cell cycle and increased cellular proliferation, and the regulation of many miRNAs in the miR-17 family in bystander tissues suggests that this may be the case. Unfortunately, due to the scarcity of tissues, an increase in cellular proliferation in this bystander model has not been assayed, although it has been found in some bystander models (12
). Future experiments in this model should assay whether cell proliferation actually occurs in bystander cells or whether the cell is just ‘poised’ to proliferate after further extracellular signals.
We can also look at these changes in cellular alignment governed by c-MYC and the miR-17 family in a different way that is more commonly associated with bystander effects. It is thought that c-MYC may prime the cell for apoptosis (45
). Taken in this context, upregulation of c-Myc in these tissues may sensitize bystander cells for an impending death signal. Indeed, we have noted a significant increase in the level of apoptotic cells in bystander issues.
Further, c-MYC and BCL2 have long been shown to associate with cancer cells, whereby it is usually the overexpression of both that leads to the cancer phenotype. The result of this co-operation is that BCL2 suppresses c-MYC-driven apoptosis (45
). However, our miRNA and protein expression patterns lead us to believe that BCL2 is downregulated via the action of miR-16. MiR-16 is significantly upregulated at 8 h.p.i., with a strong trend toward upregulation at 3 d.p.i. (). It is possible that this decrease in cellular BCL2 is functioning to accent c-MYC-directed priming of apoptosis.
Moreover, apoptosis in bystander cells is also regulated via the miR-29 family, which mediates apoptosis through the regulation of MCL1. MCL1 is a tightly controlled BCL2 family member, which is important in regulating TRAIL-mediated apoptosis (42
). MCL1 functions as a prosurvival protein by binding proapoptotic BH3-only BCL2 family members, such as BIM, BID, BIK, NOXA and PUMA (48
). Although the exact mechanisms by which the BCL2 family members mediate apoptosis is unknown, it has been shown that MCL1 binding of BID and BIM proteins protects against TRAIL-induced cell death (42
). Interestingly, it has been shown that TRAIL mediates cell–cell apoptotic RIBE (49
). This study and our previous analysis show that bystander cells exhibit increased levels of apoptosis. Furthermore, the data of this study may suggest that tumor necrosis factor-mediated cytokine signaling in RIBE may be associated with miR-29 family.
Overall, the miR-29 family plays a dual role in bystander tissues promoting both global hypomethylation through the regulation of de novo
methyltransferases DNMT3A and preparing the cell for TRAIL-mediated apoptosis through repression of anti-apoptotic MCL1. We also show that miRNA regulation in bystander tissues has been dramatically changed at 3 d.p.i. Interestingly, this corresponds to the apoptotic levels observed in this study and with the maximum apoptotic and hypomethylation levels previously seen in EpiAirway tissues under similar conditions (10
). Given that there are significant changes in miRNA expression in bystander tissues, further investigation into the role of the miR-29 family in RIBE may come to discover that this family can acts as a good biomarker for two commonly observed bystander responses.
In previous experiments (10
), there were no large-scale changes in molecular events at the 8 h.p.i. time point. This is logical, as it takes time for the bystander signal to propagate through cells and for global cellular changes to manifest. However, finding changes in miRNA expression at early time points, before the manifestation of bystander symptoms, could suggest that miRNA regulation is an upstream event of some bystander responses. Given that miRNAs may travel through gap junctions and gap junctions are necessary for bystander effects in some models (51
), one can easily envisage a future model where miRNAs from irradiated cells are quickly transported through gap junctions to prepare bystander cells for future secreted signals.
Though miRNAs mediate crucial bystander effect end points and exhibit their effects as early as 8 h.p.i., they may not necessarily be primary bystander signals. Further studies are needed to dissect the potential role of miRNAs as bystander signals and to gain further mechanistic insight into the roles of miRNAs in irradiated and bystander tissues and the potential of miRNAs to enhance or suppress the RIBE in vivo
. This challenging task can be achieved through transient transformation of irradiated and bystander cells and tissues using miRNAs or their inhibitors (antago-miRs or anti-miRs) (52
). Following the transient transformation, the impact of miRNAs or anti-miRNAs on preventing or enhancing changes in the functional RIBE readouts can be established.
Interestingly, RIBE may be mediated by reactive oxygen species (ROS) (54
). A recent study has confirmed that IR-induced oxidative stress significantly alters microRNAome of the exposed cells (55
). Furthermore, the results clearly demonstrated a common miRNA expression signature in response to radiation, hydrogen peroxide and etoposide exposure (55
). Though we have not seen a major overlap between our dataset and the reported IR- and ROS-induced miRNAs changes, some interesting parallels can be drawn. Specifically, miR-15b was upregulated by IR and ROS, whereas in our study, miR-16 was significantly upregulated in bystander tissues. These miRNAs belong to the same miRNA family and play roles in regulation of cell cycle and apoptosis (56
). Their roles in ROS-induced bystander effects and their differential regulation in responses to direct IR and RIBE need to be further analyzed.
Overall, in the future, the current study may serve as a roadmap for further understanding the mechanistic roles of miRNAs in bystander effects and for dissecting a hierarchy and cross talk between epigenetic parameters (microRNAome and DNA methylation) and well-known RIBE manifestations.