Evolutionarily conserved serum microRNAs predict radiation-induced fatality in nonhuman primates

Abstract

Effective planning for the medical response to a radiological or nuclear accident is complex. Because of limited resources for medical countermeasures, the key would be to accurately triage and identify victims most likely to benefit from treatment. We used a mouse model system to provide evidence that serum microRNAs (miRNAs) may effectively predict the impact of radiation on the long-term viability of animals. We had previously used nonhuman primates (NHPs) to demonstrate that this concept is conserved and serum miRNA signatures have the potential to serve as prediction biomarkers for radiation-induced fatality in a human population. We identified a signature of seven miRNAs that are altered by irradiation in both mice and NHPs. Genomic analysis of these conserved miRNAs revealed that there is a combination of seven transcription factors that are predicted to regulate these miRNAs in human, mice, and NHPs. Moreover, a combination of three miRNAs (miR-133b, miR-215, and miR-375) can identify, with nearly complete accuracy, NHPs exposed to radiation versus unexposed NHPs. Consistent with historical data, female macaques appeared to be more sensitive to radiation, but the difference was not significant. Sex-based stratification allowed us to identify an interaction between gender and miR-16-2 expression, which affected the outcome of radiation exposure. Moreover, we developed a classifier based on two miRNAs (miR-30a and miR-126) that can reproducibly predict radiation-induced mortality. Together, we have obtained a five-miRNA composite signature that can identify irradiated macaques and predict their probability of survival.

Fig. 1. MiRNA-based irradiation classifier development and performance

(A) Hierarchical clustering plot of miRNAs detected in the sera before and after irradiation. The heatmap depicts the 25 miRNAs that were significantly differently expressed in paired analysis of pre- (red) and post-irradiation (yellow) vehicle-treated NHPs. Red color represents high expression, blue represents low expression, and the color scale depicted above the heatmap illustrates normalized expression values with units representing standard deviation scores. Numbers above each column identify individual animal IDs, with the letters M and F representing males and females. (B) Correlation between miRNA expression differences in paired (vehicle-treated post- vs pre-irradiation) and unpaired NHPs. The expression fold differences were log2 transformed, and a value of 0 on both axes corresponds to a lack of up- or downregulation. (C)Scatterplot of three miRNAs used in the final classification model for detecting irradiation: miR-133b, miR-375, and miR-215-5p. Values are presented as dCp: differences between the average amplification cycle number in the exponential product gain phase (Cp) and the Cp of an individual miRNA, with higher values representing higher expression of miRNAs.

Fig. 2. Conservation of radiation-dependent miRNAs across the species

(A) Correlation plot of expression differences of miRNAs significantly up- or downregulated in both the NHP and mouse datasets (reanalyzed from Acharya et al. (11)). Data are presented as log2-transformed values on both axes, with a value of 0 corresponding to a lack of change. (B) Percent identity of aligned promoter sequences between Macaca mulatta (mml), Homo sapiens (hsa), and Mus musculus (mmu) for seven selected microRNAs. (C) Number of distinct transcription factors predicted to bind to the promoter region of each microRNA in each species (left Y axis, hsa - blue bars, mml - red bars, mmu - yellow bars), total number of predicted transcription factor binding sites for each microRNA in each species (right Y axis, green line), and number of overlapping transcription factors across the species for each microRNA (top table). (D) Cross-species overlap of the transcription factors with at least one binding site present in all seven microRNA promoter sequences. Seven transcription factors (listed in the text box) had binding sites in all seven microRNAs in all analyzed species.

Fig. 3. GT3 (gamma-tocotrienol) rescue’s impact on radiation-dependent miRNA expression

Expression of the seven miRNAs that showed replicable changes in both NHPs and mice depending on the time point and treatment. Different number of data points in all figures are due to undetectable amounts of particular miRNAs at individual time points. In all cases, significant differences in a post-hoc Tukey’s test are marked with asterisks: (A) miR-30a-5p, (B) miR-126-5p, (C) miR-375. (D) A scatterplot of the expression of three miRNAs (miR-30a-5p, miR-126-5p, and miR-375) grouped by treatment. MiRNA expression in GT3-treated macaques overlapped with that in pre-irradiation samples. Quantities of four miRNAs: (E) miR-133a-3p, (F) miR-215-5p, (G) miR-150-5p, (H) miR-133b showed a more extreme radiation-induced change of expression in GT3-treated macaques in comparison with that observed in vehicle-treated samples. (I) A principal component analysis plot of the three groups. The components were extracted from the seven-miRNA subset. For the purpose of this plot, missing values of the seven miRNAs were substituted with averages.

Fig. 4. Sex-specific miRNA expression and miRNA-based survival prediction

(A) Hierarchical clustering plot of miRNAs that showed significant (p<0.05) changes of expression in both paired and unpaired comparisons depending on the outcome and sex of the macaque. No clear pattern of death/survival signature was noted, with females generally dying at a higher dose than males. (B) Sex-specific differences in miR-16-2 expression relative to outcome. Expression of miR-16-2 was lower in males (red) than females (blue), but in both sexes macaques that died showed a downregulation of serum miR-16-2. Lines represent means with standard deviations. (C) Kaplan-Meier curves showing survival relative to miR-16-2 expression. Higher (>median) expression of miR-16-2 was associated with a Hazard Ratio of 0.08 (95%CI 0.01–0.69) of dying during the 60-day post-irradiation period. (D) ROC curve for the miR-30a/miR-126/sex classification model predicting the outcome of irradiation (AUC = 0.80 95%CI 0.60–1.00).

Fig. 5. Performance of the 5-miR irradiation/outcome classifier

Expression of miR-133b, miR-215, miR-375, miR-30a, and miR-126 was renormalized against the average of two selected miRNAs (miR-142 and miR-320a). (A) Scatterplot of miR-133b/miR-215/miR-375 classifier for irradiation after renormalization to miR-142 and miR-320d. (B) ROC curve showing the performance of the classifier in the validation group. The area under the ROC curve equaled 0.99 95%CI 0.98–1.00. (C) ROC curve depicting the performance of the classification model for predicting death due to irradiation in the validation group. Area under the ROC curve equaled 0.79 95%CI 0.56–1.00.