MicroRNA ssa-mir-196a-4 deceases lgr8 expression in testis development of Chinese tongue sole (Cynoglossus semilaevis)

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2022-02, Vol.258, p.110695-110695, Article 110695
• Main Authors: Tang, Lili, You, Wuxin, Wang, Qian, Huang, Fei, Shao, Changwei
• Format: Article
• Language: English
• Subjects: Animals; China; Chinese tongue sole; Fish Proteins - genetics; Fishes - genetics; In Situ Hybridization, Fluorescence; lgr8; Male; MicroRNA ssa-mir-196a-4; MicroRNAs - genetics; Receptors, G-Protein-Coupled - genetics; Testis - growth & development; Testis development
• ISSN: 1096-4959; 1879-1107
• DOI: 10.1016/j.cbpb.2021.110695

MicroRNAs (miRNAs) contribute to gonadal development in animals. However, there is little information about miRNA regulation function involved in gonadal development in fish. Our group previously identified sex-related miRNAs of Chinese tongue sole (Cynoglossus semilaevis) during sex determination and differentiation by small RNA sequencing. In the present study, we characterized ssa-mir-196a-4 and its expression in testis and verified its interaction with lgr8. miRNA ssa-mir-196a-4 precursor was predicted to have a typical hairpin structure and highly conserved among various fish species. Fluorescence in situ hybridization (FISH) of ssa-mir-196a-4 in the testis of Chinese tongue sole showed that it is mainly expressed in the cytoplasm of Sertoli cells. We determined that ssa-mir-196a-4 interacted with lgr8 by bioinformatics analysis using miRanda software. According to the dual-luciferase gene reporter assay, lgr8 is a direct target of ssa-mir-196a-4. Overexpression of ssa-mir-196a-4 in the cells of the testis cell line of Chinese tongue sole decreased the expression levels of lgr8 messenger RNA (mRNA) and protein by targeting its coding sequence (CDS) region. These results suggest that ssa-mir-196a-4 acts as a post-transcriptional regulator of lgr8 and plays an important role in developing testes of Chinese tongue sole. [Display omitted] •MicroRNA ssa-mir-196a-4 is highly conserved across various fish species.•MicroRNA ssa-mir-196a-4 was mainly expressed in the cytoplasm of testis cell of Chinese tongue sole.•The coding sequence region of lgr8 is a direct target of ssa-mir-196a-4.•The expression of lgr8 is negatively regulated by ssa-mir-196a-4.