MicroRNA Expression for Early Prediction of Late Occurring Hematologic Acute Radiation Syndrome in Baboons.

For effective medical management of radiation-exposed persons after a radiological/nuclear event, blood-based screening measures in the first few days that could predict hematologic acute radiation syndrome (HARS) are needed. For HARS severity prediction, we used microRNA (miRNA) expression changes measured on days one and two after irradiation in a baboon model. Eighteen baboons underwent different patterns of partial or total body irradiation, corresponding to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts (BCC) the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. In a two Stage study design we screened 667 miRNAs using a quantitative real-time polymerase chain reaction (qRT-PCR) platform. In Stage II we validated candidates where miRNAs had to show a similar regulation (up- or down-regulated) and a significant 2-fold miRNA expression difference over H0. Seventy-two candidate miRNAs (42 for H1-2 and 30 for H2-3) were forwarded for validation. Forty-two of the H1-2 miRNA candidates from the screening phase entered the validation step and 20 of them showed a statistically significant 2-4 fold up-regulation relative to the unexposed reference (H0). Fifteen of the 30 H2-3 miRNAs were validated in Stage II. All miRNAs appeared 2-3 fold down-regulated over H0 and allowed an almost complete separation of HARS categories; the strongest candidate, miR-342-3p, showed a sustained and 10-fold down-regulation on both days 1 and 2. In summary, our data support the medical decision making of the HARS even within the first two days after exposure where diagnostic tools for early medical decision are required but so far missing. The miRNA species identified and in particular miR-342-3p add to the previously identified mRNAs and complete the portfolio of identified mRNA and miRNA transcripts for HARS prediction and medical management.

Association of radiation-induced genes with noncancer chronic diseases in Mayak workers occupationally exposed to prolonged radiation.

We examined the association of gene expression with noncancer chronic disease outcomes in Mayak nuclear weapons plant workers who were exposed to radiation due to their occupation. We conducted a cross-sectional study with selection based on radiation exposure status of Mayak plant workers living in Ozyorsk who were alive in 2011 and either exposed to: combined incorporated Plutonium-239 ((239)Pu) and external gamma-ray exposure (n = 82); external gamma-ray exposure alone (n = 18); or were unexposed (n = 50) of Ozyorsk residents who provided community-based professional support for plant personnel and who were alive in 2011. Peripheral blood was taken and RNA was isolated and then converted into cDNA and stored at -20°C. In a previous analysis we screened the whole genome for radiation-associated candidate genes, and validated 15 mRNAs and 15 microRNAs using qRT-PCR. In the current analysis we examined the association of these genes with 15 different chronic diseases on 92 samples (47 males, 45 females). We examined the radiation-to-gene and gene-to-disease associations in statistical models stratified by gender and separately for each disease and exposure. We modeled radiation exposure as gamma or (239)Pu on both the continuous and categorical scales. Unconditional logistic regression was used to calculate odds ratios (OR), 95% confidence intervals (CI), and the concordance for genes that were significantly associated with radiation exposure and a specific disease outcome were identified. Altogether 12 mRNAs and 9 microRNAs appeared to be significantly associated with 6 diseases, including thyroid diseases (3 genes, OR: 1.2-5.1, concordance: 71-78%), atherosclerotic diseases (4 genes, OR: 2.5-10, concordance: 70-75%), kidney diseases (6 genes, OR: 1.3-8.6, concordance: 69-85%), cholelithiasis (3 genes, OR: 0.2-0.3, concordance: 74-75%), benign tumors [1 gene (AGAP4), OR: 3.7, concordance: 81%] and chronic radiation syndrome (4 genes, OR: 2.5-4.3, concordance: 70-99%). Further associations were found for systolic blood pressure (6 genes, OR: 3.7-10.6, concordance: 81-88%) and body mass index [1 gene (miR-484), OR: 3.7, concordance: 81%]. All associations were gender and exposure dependent. These findings suggest that gene expression changes observed after occupational prolonged radiation exposures may increase the risk for certain noncancer chronic diseases.

Independent validation of candidate genes identified after a whole genome screening on Mayak workers exposed to prolonged occupational radiation.

We evaluated gene expression in the peripheral blood of Mayak workers in relationship to occupational chronic exposure to identify permanent post-exposure signatures. The Mayak workers had experienced either a combined exposure to incorporated (239)Pu and external gamma rays (n = 82) or exposure to external gamma rays (n = 18). Fifty unexposed individuals served as controls. Peripheral blood was collected and then the RNA was isolated, converting it into cDNA and stored at -20°C. In a previous study at stage I, we screened the mRNA and microRNA transcriptome using 40 of the 150 samples and identified 95 mRNAs and 45 microRNAs. In stage II of this study, we now validated our 140 candidate genes using the qRT-PCR technique for the remaining 92 blood samples (18 samples were lost due to methodological reasons). We analyzed associations of normalized gene expression values in linear models separately for both exposure types (continuous and categorical scales) and adjusted for exposure age as well as stratified by gender. After further adjustment for confounders such as chronic non-cancer diseases or age at biosampling, mostly binary (on/off) dose-to-gene relationships were found for 15 mRNAs and 15 microRNAs, of which 8 mRNAs and 6 microRNAs remained significant after Bonferroni correction. Almost all of them were associated with plutonium incorporation and gender. Our study provides mRNA and microRNA gene expression changes dependent on the exposure type and gender, which occur and seem to persist after chronic radiation exposures supporting the concept of permanent post-exposure signatures.

Clinically apparent and occult metastasized seminoma: almost indistinguishable on the transcriptional level.

PURPOSE: The aim of the present study was to examine the biological differences between seminomas with occult and clinically apparent metastases at the time of diagnosis of the primary tumor to gain insight into the biology of these tumors and facilitate the identification of novel predictors of seminoma metastasis. MATERIALS AND METHODS: Total RNA including small RNAs was isolated from testicular tumors of patients with pure seminoma presenting with lymphogenic metastasis (n = 5, clinical stage IIb/c) and occult metastasis (n = 5, clinical stage I). The regulation of biological processes was examined (1) throughout the mRNA transcriptome (whole genome microarrays, 8×60 K Array, Agilent with 4 samples/group) and (2) the miRNA transcriptome employing small RNA next generation sequencing (SOLID, Life Technologies with 5 samples/group). Protein coding genes (mRNAs) and small RNAs showing a significant (≥2-fold) difference between the groups were identified. Finally (3), we examined 95 candidate miRNAs in 36 apparent metastasized and another 5 occult metastasized seminoma using logistic regression analysis. RESULTS: Among 19,596 genes, on average 12,894 mRNAs appeared expressed (65.8%, SD+/-2.4; range, 62.0-69.3%) and 16.99×106/13.94×106 small RNA reads were identified for apparent/occult metastasized seminoma. These reads on average convert into 9,901/9,675 small RNAs including 422/404 mature microRNAs. None of these mRNAs/small RNAs met our selection criteria for candidate genes. From 95 candidate miRNAs 44 appeared expressed, with 3 of them showing weak but significant (p = 0.05) differences among both groups. CONCLUSIONS: Occult and apparent metastasized seminomas are biologically almost indistinguishable and probably represent no separate tumor entities. These findings may simplify future research on seminoma metastasis.

Gene expression analysis in Mayak workers with prolonged occupational radiation exposure.

The authors evaluated gene expression in the peripheral blood in relation to occupational exposure in Mayak workers to find out about the existence of a permanent post exposure signature. Workers were exposed to combined incorporated ²³9Pu and external gamma rays (n = 82) or to external gamma rays only (n = 18), and 50 unexposed individuals served as controls. Peripheral blood was taken from workers older than 70 y. RNA was isolated, converted into cDNA, and stored at -20°C. A two-stage study design was performed focusing on examinations on the transcriptional (mRNA) and post-transcriptional level (microRNA). In the first stage, 40 samples were identified for screening purposes and selection of candidate genes. For examinations on the transcriptional level, whole genome microarrays and qRT-PCR were employed on the post-transcriptional level (667 microRNAs). Candidate genes were assessed by (1) introducing a twofold difference in gene expression over the reference group and (2) showing a significant p-value using the Kruskal-Wallis test. From 42,545 transcripts of the whole genome microarray, 376 candidate genes (80 up-regulated and 296 down-regulated relative to the reference group) were selected. Expression of almost all of these genes (70-98%) appeared significantly associated with internal ²³9Pu and to a lesser extent were associated with external gamma-ray exposure (2-30%). Associations in the same direction were found for 45 microRNAs. Although both exposures led to modulations of different gene sets in different directions, the authors could detect no differences in gene set enrichment analysis.