Changes in miRNA in the lung and whole blood after whole thorax irradiation in rats

Abstract

We used a rat model of whole thorax x-ray irradiation to profile the microRNA (miRNA) in lung and blood up to 4 weeks after radiation. MiRNA from normal and irradiated Wistar rat lungs and whole blood were analyzed by next-generation sequencing and the changes by radiation were identified by differential deRNA-seq 1, 2, 3 and 4 weeks after irradiation. The average total reads/library was 2,703,137 with a mean of 88% mapping to the rat genome. Detailed profiles of 100 of the most abundant miRNA in rat blood and lung are described. We identified upregulation of 4 miRNA, miR-144-5p, miR-144-3p, miR-142-5p and miR-19a-3p in rat blood 2 weeks after radiation that have not previously been shown to be altered after radiation to the lung. Ingenuity Pathway Analysis identified signaling of inflammatory response pathways. These findings will support development of early detection methods, as well as mechanism(s) of injury and mitigation in patients after radiotherapy or radiological accidents.

Figure 1

The top 57 most abundant mature miRNAs in the blood (a) and lung (b) of normal rats.

Figure 2. Relative miRNA expression after 2 weeks post-radiation in blood, as determined by RT-qPCR.

The line in each box represents the median value. The box spans the 25th and 75th percentiles. The upper and lower bars show the maximum and minimum values. N = 11 rats/box. *P < 0.05 radiated vs. control (0 Gy). MiR-191-5p was used as reference because it shows consistent read counts in all sequenced libraries. miRNA in (a) were selected by results of miRNA-seq for candidates with p < 0.05 radiated vs. control by t test. miRNA in (b) were selected because other investigators previously identified these to be altered after radiation or lung injury. Their reads count with p values from the sequence results of this study are shown in (c). # refers to the library number # in Table 1.

Figure 3

Blood cell counts versus time are shown (a) White blood cell count (WBC) and (b) Red blood cell count (RBC). *p < 0.05 vs. control (0 Gy). N = 14 (controls), 6 (irradiated at 1 week), 7 (irradiated at 2 weeks), 9 (irradiated at 3 weeks), 9 (irradiated at 4 weeks). The line represents the median.

Figure 4. Ingenuity pathway analysis of the sequence data showed the top scoring diseases and function that are associated with our model (upper panel).

The pathway map is presented below and miRNA that were significantly changed in the miRNA-seq study and also validated by qPCR are shown as highlighted in red.