Spaceflight alters expression of microRNA during T-cell activation

Abstract

Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.

Keywords: epigenetics; gene expression; immune response; microgravity.

Figure 1.

Heat map of predicted miR-21 target genes showing differentially regulated 1.5 h after T-cell activation in microgravity (μg) and normal gravity (1g). Genes were grouped based on expression profile using Pearson's centered metric with centroid linkage. Rows represent gene probes; columns represent expression profiles of donors. Heat map scale ranges from green (down-regulation) to red (up-regulation) for log₂ normalized expression values. Blue-labeled columns are normal gravity-activated samples, red-labeled columns are microgravity-activated samples, and yellow-labeled columns are microgravity-nonactivated T cells. A total of 17 unique miR-21 gene targets are shown with differential expression between normal gravity and microgravity condition (P ≤ 0.05; FASLG P ≤ 0.06). There is considerable variability between donors in nonactivated samples and some variability in microgravity-activated donors. Gene expression in normal gravity-activated samples becomes considerably more uniform 1.5 h after activation.

Figure 2.

Promoter regions of IL-1, IL-2-Rα, Premir-21, TAGAP, SPRY2, and FASLG are induced by AP1 and/or NF-κB-1α; 2 of transcription factors in early gene expression (these 2 transcription factors are found in promoter region of nearly 40% of T-cell early induced genes).

Figure 3.

Real-time confirmation of miR-21 down-regulation of gene expression in CD4⁺ T cells flown on ISS. Cells were placed in microgravity (μg), 0.5g, or normal gravity (1g) for 3 h before activation. At 1.5 h after activation with concanavalin A/anti-CD28, RNA was stabilized in RNALater. Total RNA was isolated, and miR-21 expression was quantified using mirVana miRNA detection kit. Expression was corrected to internal standard 5S rRNA. Bars represent mean ± sd of independent biologic replicates (n = 6). *P < 0.05; **P < 0.001 with 2-tailed Student t test against activated microgravity samples (μg Act).

Figure 4.

qRT-PCR of genes selected from gene array analysis in nonactivated and concanavalin A/anti-CD28 activated human lymphocytes in flown normal gravity-activated and flown microgravity conditions indicating altered gene expression in ISS spaceflight vs. normal gravity onboard controls (1.5 h). We used qRT-PCR to analyze mir-21 targets BTG2, TAGAP, SPRY2, and FASLG in ISS samples that were activated and fixed in orbit. NFKBIA and CD40LG were also analyzed because they are important in T-cell activation.

Figure 5.

Postulated actions of miR-21 and gene expression of early T-cell activation. Hypothesized pathways of expression and down-regulation. miR-21 is known to down-regulate AP1 (28), and from presence of its seed sequence in 3′-UTR of target genes miR-21 is predicted to down-regulate 4 targets approximately 24 h after activation; BTG2, TAGAP, SPRY2, and FASLG as miR-21 expression increases. It is possible that miR-21 provides a mechanism of self-limited induction regulating T-cell activation.