Identification and Investigation of miRNAs From Gastrodia elata Blume and Their Potential Function

Abstract

Gastrodia elata Blume (G. elata) is a valuable traditional Chinese medicine with neuroprotection, anti-inflammatory, and immune regulatory functions. MicroRNAs (miRNA) is a kind of endogenous noncoding small RNAs that plays distinctly important roles for gene regulation of organisms. So far, the research on G. elata is mainly focused on the pharmacological functions of the natural chemical ingredients, and the function of G. elata miRNA remains unknown. In this study, 5,718 known miRNAs and 38 novel miRNAs were identified by high-throughput sequencing from G. elata. Based on GO and KEGG analysis, we found that the human genes possibly regulated by G. elata miRNAs were related to the cell cycle, immune regulation, intercellular communication, etc. Furthermore, two novel miRNAs as Gas-miR01 and Gas-miR02 have stable and high expression in the medicinal tissues of G. elata. Further bioinformatics prediction showed that both Gas-miR01 and Gas-miR02 could target Homo sapiens A20 gene, furthermore, the dual-luciferase reporter gene assay and Western Blotting verified the interaction of Gas-miR01 or Gas-miR02 with A20. These evidences suggested that G. elata-unique miRNAs might be involved in certain physiological processes. The animal experiment showed that Gas-miR01 and Gas-miR02 could be detected in some tissues of mice by intragastric administration; meanwhile, the A20 expression in some tissues of mice was downregulated. These results supported for the functional study of G. elata miRNAs.

Keywords: A20; Gastrodia elata Blume; expression profiles; functional study; high-throughput sequencing; microRNA.

Figure 1

The life cycle of G. elata (trace diagram). The life cycle of G. elata has five stages, including G. elata seeds, protocorms, white G. elata, arrow G. elata, and bolted G. elata.

Figure 2

Length distribution of G. elata sRNA and expression profiles of 10 G. elata miRNAs in different tissues. Length distributions of clean total reads (A) and clean unique reads (B). (C) Clustering map of difference miRNAs. (D) Relative expression levels of 10 G. elata miRNAs in different tissues.

Figure 3

Expression profiles of 10 G. elata miRNAs in different growth stages. The relative G. elata miRNA expression levels in different growth stages were determined by qRT-PCR. The vertical coordinate means fold change.

Figure 4

GO and KEGG enrichment analysis of differential miRNA target genes. (A) The target genes of miRNA involve BP (biological processes), CC (cellular components), and MF (molecular functions). (B) The closer the Q-value is to zero, the more significant the particular GO term associated with the group of genes; the closer the Q-value is to one, the opposite. The closer the Q-value is to 0, the more significant the particular GO term associated with the group of genes; the closer the Q-value is to one, the opposite.

Figure 5

The down-regulation of A20 by Gas-miR01 and Gas-miR02. (A) Matching between Gas-miR01, Gas-miR02 and their target sites in A20 gene. (B) The relative luciferase activity down-regulated by Gas-miR01. (C) The relative luciferase activity down-regulated by Gas-miR02. (D) Levels of Gas-miR01 and Gas-miR02 in 293T cells after transfected with corresponding mimics. (E) The expression level of A20 protein after transfected with Gas-miR01 and Gas-miR02 mimics. *p < 0.05; **p < 0.01; ***p < 0.001. Values are the mean ± SEM.

Figure 6

Gas-miR01 and Gas-miR02 were detected in some tissues of mice by intragastric administration. (A) Flow chart of animal experiments. The levels of Gas-miR01 (B) and Gas-miR02 (C) in fresh G. elata tuber, Gastrodia decoction and Gastrodia powder. The fresh G. elata total RNAs, Gastrodia decoction and Gastrodia powder were gavaged and the levels of Gas-miR01 and Gas-miR02 in various tissues of mice were detected by qRT-PCR. The levels of Gas-miR01 and Gas-miR02 in mouse tissues. (D) cerebrum; (E) cerebellum; (F) hypothalamus; (G) heart; (H) kidney; (I) spleen. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control group, n = 6. Values are the mean ± SEM.

Figure 7

The A20 expression was downregulated in many tissues of mouse. The result of qRT-PCR showed the mRNA level of A20 in various tissues of mice. (A) Cerebrum. (B) Cerebellum. (C) Hypothalamus. (D) Heart. (E) Kidney. (F) Spleen. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control group, n = 6. Values are the mean ± SEM.