De novo transcriptome analysis and gene expression profiling of fish scales isolated from Carassius auratus during space flight: Impact of melatonin on gene expression in response to space radiation

Abstract

Astronauts are inevitably exposed to two major risks during space flight, microgravity and radiation. Exposure to microgravity has been discovered to lead to rapid and vigorous bone loss due to elevated osteoclastic activity. In addition, long‑term exposure to low‑dose‑rate space radiation was identified to promote DNA damage accumulation that triggered chronic inflammation, resulting in an increased risk for bone marrow suppression and carcinogenesis. In our previous study, melatonin, a hormone known to regulate the sleep‑wake cycle, upregulated calcitonin expression levels and downregulated receptor activator of nuclear factor‑κB ligand expression levels, leading to improved osteoclastic activity in a fish scale model. These results indicated that melatonin may represent a potential drug or lead compound for the prevention of bone loss under microgravity conditions. However, it is unclear whether melatonin affects the biological response induced by space radiation. The aim of the present study was to evaluate the effect of melatonin on the expression levels of genes responsive to space radiation. In the present study, to support the previous data regarding de novo transcriptome analysis of goldfish scales, a detailed and improved experimental method (e.g., PCR duplicate removal followed by de novo assembly, global normalization and calculation of statistical significance) was applied for the analysis. In addition, the transcriptome data were analyzed via global normalization, functional categorization and gene network construction to determine the impact of melatonin on gene expression levels in irradiated fish scales cultured in space. The results of the present study demonstrated that melatonin treatment counteracted microgravity‑ and radiation‑induced alterations in the expression levels of genes associated with DNA replication, DNA repair, proliferation, cell death and survival. Thus, it was concluded that melatonin may promote cell survival and ensure normal cell proliferation in cells exposed to space radiation.

Keywords: space radiation; transcriptome; de novo assembly; fish scale; gene network; melatonin.

Figure 1.

Workflow of de novo transcriptome analysis in goldfish scales. DEGs, differentially expressed genes; TMM, Trimmed Mean of M value.

Figure 2.

PCA of the comprehensive gene expression data. PCA was performed on data from the G, F-µG, F-1G, G + melatonin and F-µG + melatonin groups. Fish scales isolated from Carassius auratus were cultured on ground condition or space flight in the presence or absence of microgravity/melatonin until freezing, followed by RNA extraction and transcriptome analysis. The data represents a single sequencing experiment of biological triplicates. PCA, principal component analysis; G, ground samples; F-µG, space flight samples in the presence of microgravity; F-1G space flight samples in the absence of microgravity.

Figure 3.

Venn diagrams and hierarchical clustering of identified differentially expressed transcripts in goldfish scales. (A) Venn diagrams of upregulated and downregulated transcripts between the G group and space flight samples F-µG or F-1G. (B) Heatmaps of the differentially expressed transcripts identified from G, F-µG, F-1G, G + melatonin and F-µG + melatonin groups. The color spectrum (green to orange) represents the log2 expression values scaled between-0.66 and 0.66 (low to high expression). G, ground samples; F-µG, space flight samples in the presence of microgravity; F-1G space flight samples in the absence of microgravity.

Figure 4.

Molecular and cellular function analysis of genes responsive to melatonin. Top five molecular and cellular functions of upregulated and downregulated genes based on P-values calculated by Ingenuity Pathway Analysis.

Figure 5.

Gene networks identified using differentially expressed gene analysis. (A) Upregulated and (B) downregulated genes following melatonin treatment were analyzed using Ingenuity Pathway Analysis tools. Red nodes represent upregulated genes, while the green nodes are for downregulated genes. Solid lines and dashed lines indicate direct and indirect interactions between molecules, respectively. (C) Heatmap of genes in the identified networks based on relative expression values. The green to red color spectrum represents the log2 ratios scaled between-0.66 and 0.66 (low to high expression). G, ground samples; F-µG, space flight samples with microgravity; F-1G space flight samples with 1G. The expression value and gene names are also shown in Table SII. EGFR, epidermal growth factor receptor; ENO1, enolase 1; RHOA, Ras homologue family member A; HIPK2, homeodomain-interacting protein kinase 2; INHBA, inhibin subunit β A; LGR4, leucine-rich repeat-containing G protein-coupled receptor 4; Sox6, SRY-box transcription factor 6.