EXPRESSION OF MIR-202-5P IN GONADS AND VERIFICATION OF TARGETING RELATIONSHIP BETWEEN MIR-202-5P AND CBX2 IN PARALICHTHYS OLIVACEUS

Gonadal development is a complex and essential process for the organisms with sexual reproduction. With over 30000 known species, fish constitute the largest and most diverse taxa, and display almost all reproductive strategies of vertebrates. In fish, generally, sex determination first determines the direction of gonadal development. On the basis of gonadal differentiation, individual gonad develops into mature ovary and testis through complex interactions of genetic, environmental, physiological and other factors. The whole developmental process is driven sequentially by the stage-specific expression of a series of genes at both transcriptional and post-transcriptional levels, and thus gonadal development involves the expression of numerous mRNAs and microRNAs (miRNAs). MiRNAs are a typical group of non-coding single-stranded small RNAs containing 21—23 nucleic acids. The mature miRNAs usually interact with the 3′-untranslated region (3′UTR) of target mRNA in the form of complementary base pairs, thus negatively regulating the gene expression in the post-transcriptional stage by mRNA degradation and translational inhibition. Increasing evidence reveals that miRNAs are abundant in male gonad and play a crucial part in gonadal development. A series of miRNAs related to gonadal development have been successively discovered, and among these miRNAs, miR-202-5p is considered to be a miRNA specifically expressed in vertebrate gonads, but the study on its target genes and functions in fish is still very limited, and the function and regulatory mechanism of miR-202-5p in gonadal development remain unclear. The Japanese flounder (Paralichthys olivaceus) is an important marine economic fish. It displays significant sexual dimorphism in growth trait between male and female individuals, with females having a faster growth rate and higher economic value than males. The expression profiles of miRNAs or mRNAs in bisexual gonads have been reported in Japanese flounder. We have established the miRNA libraries derived from Japanese flounder testis and ovary, finding that miR-202-5p has significantly differential expression in bisexual gonads. In this study, the expression profile of miR-202-5p in different tissues and bisexual gonads of Japanese flounder was constructed by using real-time PCR and then the co-localization analysis of miR-202-5p with the germline cell marker vasa gene was established by in situ hybridization. The real-time PCR results showed that miR-202-5p was specifically expressed in the gonads, and its expression in the testis was higher than that in the ovary. In order to further detect the localization and expression of miR-202-5p in the gonads, HE and in situ hybridization analysis were performed in this study, and the germline cell marker gene vasa was used as a localization reference. The results of in situ hybridization revealed that miR-202-5p was mainly expressed in spermatogonia and spermatocytes of the testis, but only in the stage IV and V oocytes of the ovary. Importantly, the overlapping distribution of miR-202-5p and vasa in Japanese flounder better revealed its cellular location, implying that miR-202-5p should be a very important specific miRNA during gonadal development. The latest research has found that chromobox homolog 2 (CBX2) plays an important role in gonadal development. PcG is a conserved protein family, which mainly targets at a series of genes related to cell differentiation and development, and carries out epigenetic modification to achieve gene silencing at the chromatin level. After the mutation of cbx2, it can not be properly combined with the downstream target genes and will bind to different sequences, thus losing the regulatory effect on the downstream target genes such as sf1, and thus unable to fully regulate the expression of the target genes necessary for gonadal development, thus causing the failure of normal gonadal development or sexual inversion. The prediction and identification of miRNA target genes is the key basis for studying miRNA functions. The dual luciferase reporter gene detection technology is highly sensitive and easy to operate. It is the most commonly used method to identify miRNA target genes currently. To further explore the function of miR-202-5p in gonadal development of Japanese flounder, the targeting relationship between miR-202-5p and cbx2 was identified by bioinformatics method prediction and dual luciferase reporter gene technology, and the results confirmed that cbx2 is a target gene directly regulated by miR-202-5p. In summary, miR-202-5p was expressed in a gonad-specific and sex-biased manner in Japanese flounder. It predominately localized in spermatogonia and spermatocyte. Based on bioinformatics analysis and luciferase assay verification, cbx2 was confirmed as a direct regulatory target of miR-202-5p in vitro. This study provides a basis for further elaborating the mechanism of miR-202-5p in gonadal development of Japanese flounder. However, it is necessary to conduct further experiments to discover other targets and elucidate the regulatory mechanism of miR-202-5p in fish gonadal development.