The Heterogeneous Nuclear Ribonucleoprotein K (hnrnpk) Gene Targeted by miR-460a-5p Functions in the Gonadal Differentiation and Development in Chinese Tongue Sole (Cynoglossus semilaevis)

Chinese tongue sole (Cynoglossus semilaevis), an economically important mariculture species in China, exhibits pronounced sexual dimorphism in growth, underscoring the importance of elucidating sex regulatory mechanisms for aquaculture development. Heterogeneous nuclear ribonucleoprotein K (hnrnpk) critically regulates mammalian reproductive development, yet its role in fish sex regulation remains elusive. Here, we systematically investigated the underlying function and mechanisms of hnrnpk in C. semilaevis through integrated molecular cloning, expression profiling, upstream regulatory analysis, functional assays, and transcriptome sequencing. We found that hnrnpk was highly expressed in the gonad and liver, with female-biased expression during gonadal development. Promoter activity assays revealed that sox2 and c-Jun enhanced hnrnpk transcription, whereas foxl2 and ar suppressed it. Additionally, hnrnpk was directly targeted by miR-460a-5p in C. semilaevis, revealing multi-level transcriptional and post-transcriptional regulation. Functional analyses showed that hnrnpk regulated cyp19a1a in a cell type-dependent and dose-sensitive manner: the expression of cyp19a1a was both upregulated in hnrnpk-knockdown ovarian cells and hnrnpk-overexpression testicular cells. Interestingly, foxl2 was upregulated in hnrnpk-knockdown ovarian cells but suppressed in hnrnpk-overexpression testicular cells, which showed the distinct regulation mechanisms in the different sexual programs. Transcriptomic analyses further revealed that several sex-related genes (sox9a with downregulation, etc.) were significantly regulated, and cell development and cycle pathways were dramatically enriched in functional enrichment analyses. This might indicate that hnrnpk overexpression drives C. semilaevis testis (CSTE) toward feminization reprogramming through sox9 switching and multi-pathway perturbations. Overall, our findings might reveal that hnrnpk, a female-biased gene regulated by miR-460a-5p and transcription factors, influences sex-related gene expression through sox9 switching. This study will offer new insights for C. semilaevis hnrnpk into sex determination and also provide a potential target for monosex breeding in aquaculture.