Research ArticleRADIATION INJURY

Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury

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Science Translational Medicine  13 May 2015:
Vol. 7, Issue 287, pp. 287ra69
DOI: 10.1126/scitranslmed.aaa6593
  • Fig. 1. Characterization of hematopoietic injury in C57BL/6J mice after exposure to TBI.

    (A) Establishment of sublethal and lethal dose in C57BL/6J mice. Kaplan-Meier survival curves of C57BL/6J male mice exposed to 0 (control), 2, 6.5, or 8 Gy of TBI (n = 20 per group). P value determined by log-rank (Mantel-Cox) test. (B to E) C57BL/6J mice were exposed to TBI at the indicated doses and allowed to recover for up to 3 months (B). At each time point, animals were sacrificed and bone marrow was analyzed for number of BM-MNCs (C), CFU-C content (D), and frequency of LKS HPCs (E) per hind limb. Data in (C) and (D) are means ± SEM (n = 5 per group; two independent experiments). Data in (E) are individual animals, and horizontal bars are means. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant; one-way analysis of variance (ANOVA) followed by Tukey’s test.

  • Fig. 2. Stem cell transplantation from irradiated mice reveals a defect in short- and long-term repopulating ability.

    (A) Total number of HSCs (LKS+ cells) per hind limb measured by FACS. Data are individual animals, and horizontal bars are means. (B) Schematic to describe transplantation (Tx) of HSCs or unfractionated whole bone marrow (WBM) into lethally irradiated recipients. BM, bone marrow. (C) Representative FACS profiles of stained bone marrow from control and irradiated donor mice used to sort HSCs for transplant. Individual profiles show total scatter, lineage (lin), and LKS+ gates. FSC, forward-scattered light; SSC, side-scattered light. (D) Donor cell engraftment in peripheral blood of recipients transplanted with either HSCs or WBM. Total leukocyte engraftment at 1 and 4 months posttransplant is shown. Data are means ± SEM (n = 5 per group for HSC, n = 4 per group for WBM). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant; compared to control (0 Gy) mice; one-way ANOVA followed by Tukey’s test.

  • Fig. 3. Serum miRNA profiling and identification of radiation dose–specific miRNA signatures.

    C57BL/6J mice were exposed to 0, 2, 6.5, or 8 Gy of TBI (n = 10 per group). Serum collected from these animals 24 hours after TBI was subjected to miRNA profiling. Signatures consisting of the most highly altered five miRNAs were generated. (A) Control (0 Gy) versus 2-Gy miRNA signature with hierarchical clustering depicting relationship between individual samples. (B) Validation of the 0-Gy versus 2-Gy signature in (A) with an independent set of animals. (C) miRNA fold changes in 2-Gy irradiated animals compared to 0-Gy controls at 24 hours and 7 days after TBI. (D) Signature for 2 Gy versus 6.5 Gy with hierarchical clustering depicting relationship between individual samples. (E) Validation of the 2-Gy versus 6.5-Gy signature in (D) with an independent set of animals. (F) miRNA fold changes in 6.5-Gy irradiated animals compared to 2 Gy at 24 hours and 7 days after TBI. Data in (B), (C), (E), and (F) are normalized to miR-101a or miR-19b. Data in (B) and (E) are individual animals with means ± SEM (n = 6 to 10 per group, two independent experiments). Data in (C) and (F) are means ± SEM (n = 4 to 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant; paired t test.

  • Fig. 4. An miRNA signature can differentiate between sublethal and lethal radiation exposure.

    (A) The 6.5-Gy versus 8-Gy miRNA signature presented with hierarchical clustering showing relationship between individual samples. (B) Validation of the 6.5-Gy versus 8-Gy signature in (A) with an independent set of animals. Data are individual animals with means normalized to miR-101a ± SEM (n = 7 to 9 per group, two independent experiments). (C) miRNA fold changes in 8-Gy irradiated animals compared to 6.5 Gy at 24 hours, 3 days, and 7 days after TBI. Data in (C) are means normalized to miR-101a ± SEM (n = 4 to 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant; paired t test.

  • Fig. 5. The sublethal versus lethal miRNA signature predicts impact of the radioprotective agents.

    (A) Schematic of experiment with C57BL/6J mice given amifostine (ami) or saline (sal) 1 hour before 0 or 8.5 Gy TBI (n = 10 per group). Serum was isolated for miRNA profiling 24 hours later. (B) Kaplan-Meier survival curves of mice. P value determined by log-rank (Mantel-Cox) test. (C) Relative levels of indicated miRNAs in the sera of mice. Data are individual animals with means normalized to miR-101a ± SEM (n = 5 per group; two independent experiments). (D) Correlation of relative expression ratios of miRNAs in the 6.5-Gy versus 8-Gy signature from two separate experiments: those described in (A) and in Fig. 4A (r = 0.97; P = 0.0067, Pearson’s correlation). **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant; one-way ANOVA followed by Dunnett’s test.

  • Fig. 6. The sublethal versus lethal miRNA signature predicts impact of radiomitigating agents.

    (A) Survival curve of C57BL/6J mice exposed to 10.4 Gy of TBI followed by transplantation of BMSCs (n = 5 per group). P value determined by log-rank (Mantel-Cox) test. (B) Fold change of indicated miRNAs from sera of animals in (A) at day 5 after TBI. Data are means normalized to miR-101a ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; one-way ANOVA followed by Dunnett’s test.

  • Fig. 7. Sublethal versus lethal miRNA signature correlates with the protective effect of amifostine in NSG mice engrafted with human CD34+ HSCs.

    (A) Representative (n = 5) FACS plots showing percent engraftment of human CD45+ cells in the bone marrow and peripheral blood of NSG mice transplanted with human CD34+ HSCs. (B to E) Humanized NSG mice were pretreated with amifostine or saline and exposed to 4.5 Gy of TBI. Control animals received no pretreatment and 0 Gy of irradiation. Moribund animals were analyzed for total bone marrow cellularity (B), number of human CD45+ cells per hind limb (C), and CFU-Cs measured at 7 days after plating in human methylcellulose media (D). Data are individual animals with means ± SEM (n = 5 to 8 per group). Relative miRNA levels in the sera of humanized mice at 24 hours after exposure to TBI (E). Data are individual animals with means normalized to miR-101a ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant, by paired t test (B to D) or one-way ANOVA followed by Dunnett’s test (E).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/287/287ra69/DC1

    Fig. S1. Analysis of peripheral blood CBC parameters after TBI-induced hematopoietic damage.

    Fig. S2. Repopulation analysis at 1 month after HSC transplant.

    Fig. S3. Repopulation analysis at 4 months after HSC transplant.

    Fig. S4. Repopulation analysis after unfractionated whole bone marrow transplant.

    Fig. S5. Serum miRNA profiling.

    Fig. S6. miRNAs significantly altered in all irradiated samples.

    Fig. S7. Radiation dose dependence of select miRNAs.

    Fig. S8. Identical mature miRNA sequences in human and mouse.

    Table S1. Peripheral blood CBC levels in individual mice.

    Table S2. Fold changes of 68 statistically significant miRNAs at different doses of TBI with respect to controls.

    Table S3. Significantly altered miRNAs between 6.5 and 8 Gy of irradiation and pretreatment with amifostine.

    Table S4. miRNAs in the 6.5-Gy versus 8-Gy signature as indicators of survival after TBI.

    Table S5. Percent human CD45+ cell engraftment in individual huCD34+ NSG (humanized) mice.

    Table S6. Peripheral blood CBC in individual huCD34+ NSG (humanized) mice.

    Table S7. Target sequences of individual miRNAs.

    Reference (38)

  • Supplementary Material for:

    Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury

    Sanket S. Acharya, Wojciech Fendler, Jacqueline Watson, Abigail Hamilton, Yunfeng Pan, Emily Gaudiano, Patryk Moskwa, Payel Bhanja, Subhrajit Saha, Chandan Guha, Kalindi Parmar, Dipanjan Chowdhury*

    *Corresponding author. E-mail: dipanjan_chowdhury{at}dfci.harvard.edu

    Published 13 May 2015, Sci. Transl. Med. 7, 287ra69 (2015)
    DOI: 10.1126/scitranslmed.aaa6593

    This PDF file includes:

    • Fig. S1. Analysis of peripheral blood CBC parameters after TBI-induced hematopoietic damage.
    • Fig. S2. Repopulation analysis at 1 month after HSC transplant.
    • Fig. S3. Repopulation analysis at 4 months after HSC transplant.
    • Fig. S4. Repopulation analysis after unfractionated whole bone marrow transplant.
    • Fig. S5. Serum miRNA profiling.
    • Fig.S6. miRNAs significantly altered in all irradiated samples.
    • Fig. S7. Radiation dose dependence of select miRNAs.
    • Fig. S8. Identical mature miRNA sequences in human and mouse.
    • Table S1. Peripheral blood CBC levels in individual mice.
    • Table S2. Fold changes of 68 statistically significant miRNAs at different doses of TBI with respect to controls.
    • Table S3. Significantly altered miRNAs between 6.5 and 8 Gy of irradiation and pretreatment with amifostine.
    • Table S4. miRNAs in the 6.5-Gy versus 8-Gy signature as indicators of survival after TBI.
    • Table S5. Percent human CD45+ cell engraftment in individual huCD34+ NSG (humanized) mice.
    • Table S6. Peripheral blood CBC in individual huCD34+ NSG (humanized) mice.
    • Table S7. Target sequences of individual miRNAs.
    • Reference (38)

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