Dysregulation of lncRNA and circRNA Expression in Mouse Testes after Exposure to Triptolide

Abstract

Background: Triptolide has been shown to exert various pharmacological effects on systemic autoimmune diseases and cancers. However, its severe toxicity, especially reproductive toxicity, prevents its widespread clinical use for people with fertility needs. Noncoding RNAs including lncRNAs and circRNAs are novel regulatory molecules that mediate a wide variety of physiological activities; they are crucial for spermatogenesis and their dysregulation might cause male infertility. However, whether they are involved in triptolide-induced reproductive toxicity is completely unknown.

Methods: After exposure of mice to triptolide, the total RNAs were used to investigate lncRNA/circRNA/mRNA expression profiles by strand-specific RNA sequencing at the transcriptome level to help uncover RNA-related mechanisms in triptolide-induced toxicity.

Results: Triptolide significantly decreased testicular weight, damaged testis and sperm morphology, and reduced sperm motility and density. Remarkable deformities in sperm head and tail were also found in triptolide-exposed mice. At the transcriptome level, the triptolide-treated mice exhibited aberrant expression profiles of lncRNAs/circRNAs/mRNAs. Gene Ontology and pathway analyses revealed that the functions of the differentially expressed lncRNA targets, circRNA cognate genes, and mRNAs were closely linked to many processes involved in spermatogenesis. In addition, some lncRNAs/circRNAs were greatly upregulated or inducibly expressed, implying their potential value as candidate markers for triptolide-induced male reproductive toxicity.

Conclusion: This study provides a preliminary database of triptolide-induced transcriptome, promotes understanding of the reproductive toxicity of triptolide, and highlights the need for research on increasing the medical efficacy of triptolide and decreasing its toxicity.

Keywords: RNA sequencing; Triptolide; circRNA; lncRNA; male infertility; spermatogenesis..

Fig. (1)

Effect of triptolide on testis and sperm morphology and physiological sperm parameters. The mice received either physiological saline solution (control) or administered triptolide with i.g. of 50 μg/kg Body weight/day. Mice were euthanized by cervical dislocation at 35 days after treatment. Testes and spermatozoa from the cauda epididymis were harvested quickly. After testis sections were stained with hematoxylin and eosin (H&E), testis morphology (A) (B) was observed under optical microscope. Body (C) and testicular weights (D) were measured using an electronic balance. Sperm morphology (E) (F) was also observed under optical microscope. Sperm abnormality rate (G) Sperm motility (H), and sperm density (I) were analyzed using a CASA system. At least 200 spermatozoa were counted for each assay. Data are presented as the mean ± SE, (n = 6). **P < 0.01 compared with control.

Fig. (2)

lncRNA/cicrRNA/mRNA expression profiles after triptolide exposure. The differentially expressed genes were analyzed using DEGseq software based on the FPKM method (≥ 2.0-fold-change with P < 0.05). The number of differentially expressed genes (A). Differentially expressed lncRNAs (B), cicrRNAs (C) and mRNAs (D) in testes.

Fig. (3)

Validation of sequencing data using qPCR. Total RNA was isolated from testes after exposure of mice to triptolide for 35 days, and then qPCR was performed to detect the RNA expression levels. β-actin gene was used as loading control to normalize RNA expression levels. Data are expressed as the mean ± SE (n = 3).