Low-Dose Ionizing Radiation Modulates Expression of Gpam in Immortalized EBV Infected B Cells through miRNA Network

Low-dose ionizing radiation (LDIR, ≤ 0.1 Gy) which is typically associated with therapeutic and diagnostic radiological modalities, is known to induce remodeling of the stromal microenvironment. Whereas the biologic responses to LDIR are commonly described as a stress response, the carcinogenic potential of this environmental stressor remains unknown. Recently, ionizing radiation (IR)-induced alterations of miRNA expression that play a fundamental role in cell signaling events, have been demonstrated in various cell types (Marsit, Cancer Res. 2006).

To assess a potential determinant influencing of LDIR induced miRNA alterations in pre-malignant cells in a microenvironment, we utilized immortalized pre-malignant Epstein-Barr virus infected-B (EBV-B) cells which are continuously proliferating in circulation or in lymph nodes. The LDIR system (0.1 Gy, 4MV X ray from a LINAC) was utilized in the in vitro co-culture of EBV-B and mesenchymal stromal cells (MSC) to mimic the lymph node stromal microenvironment. We confirmed that MSC protected co-cultured EBV-B cells from spontaneous apoptosis and caused accumulation of EBV-B cells in the G0/G1 phase (EBV-B; monoculture vs coculture with MSC; Sub G1% 42.0±2.4 vs 34.1±0.9 p< 0.01, G0/G1 % 42.6±2.1 vs 47.6±2.0, p<0.05).

To identify LDIR-induced modulation of specific miRNAs in EBV-B cells, a high-confidence list of 44 known LDIR-associated miRNAs from three separate screening studies of lymphoblastic cells was assembled (Cha et al., Oncol Rep.2009, Chaudhry et al., J Biomed Sci. 2010, Lhakhang et al., Comp Funct Genomics. 2012). From these miRNA, we focused on four miRNAs let-7a, miR-16, miR-19b and miR-21 that have been listed in more than two of the screens. Our studies showed that LDIR upregulated let-7a and miR-16 levels in irradiated mono-cultured EBV-B, but on the contrary downregulated all tested miRNAs in MSC co-cultured EBV-B cells (at 24-hour).

We next investigated alterations of EBV-B gene expression by LDIR using the DNA microarray (Affymetrix). In mono-cultured EBV-B cells, cDNA microarray analysis detected upregulation of TGFB1 mRNA, and downregulation of the genes encoding a lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase (GPAM), a growth factor amphiregulin (AREG), cell survival-/growth-related factors Rho-GTPase effector gene folmin-1 (FMN1), chemotaxis-inducing chemokine gene IL-8 and B-cell lymphoma 2 gene BCL2, all of which were confirmed by qRT-PCR. Downregulation of the GPAM protein was further shown on the protein level by western blot, and the ontology analysis demonstrated that LDIR caused the TGFbeta-dependent induction of the epithelial-mesenchymal transition (EMT) pathway in mono-cultured EBV-B cells. In contrast to the mono-culture condition, the GPAM mRNA and protein expression were upregulated in MSC co-cultured EBV-B cells.

To determine the potential targets of LDIR-altered miRNAs, we next identified 79 genes that are commonly targeted by let-7a, miR-16, miR-19b, or miR-21 from a microrna.org database, and found that the expression changes of GPAM mRNA and protein were strikingly matched with miRNA profiling. Partial concordant changes of DNAJ(HSP40)A2 coding heat shock protein (HSP) 40 homolog, a cochaperone of HSP70s, CPEB3 coding RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CEBP3) and its transcriptional target GLUR2 (glutamate receptor subunit), a receptor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were also observed in the microarray results.

Since GPAM is a lipid-related metabolism gene which is known to be regulated by miR-27b and to negatively impact survival of breast cancer cells we next investigated the expression changes of miR-27b after LDIR in EBV-B cells. As expected, LDIR upregulated miR-27b in mono-cultured EBV-B cells, and downregulated in co-cultured EBV-B cells with MSC. Intriguingly, no significant changes of GPAM mRNA as well as tested miRNA expression was observed by 1Gy irradiation.

In summary, we demonstrated that LDIR directly modulates EBV-B cells gene expression. Importantly, LDIR additionally impacts EBV-B cells indirectly through miRNA modulation by the neighboring stromal cells.