Identification of the Hc-inhba gene and its regulation by miR-9-5p in Hyriopsis cumingii

Inhibin subunit beta A (inhba) encodes the inhibin subunit beta A chain and plays an important regulatory role in animal reproductive function. To understand the role of inhba in the Hyriopsis cumingii, the full-length cDNA sequence of Hc-inhba was cloned using rapid amplification of cDNA ends (RACE). Real-time quantitative PCR (RT-qPCR) was performed to analyze the expression patterns of Hc-inhba mRNA in different tissues and during early gonadal development in H. cumingii. RNA interference (RNAi) was employed to knock down Hc-inhba expression, and the resulting changes in the expression of related genes were examined. Additionally, dual-luciferase reporter assays and miR-9-5p antagomir injection experiments were conducted to validate the targeting regulation of Hc-inhba by miR-9-5p both in vitro and in vivo, we obtained a full-length cDNA of Hc-inhba from the H. cumingii, which contains a 241 bp 5′ untranslated region, a 670 bp 3′ untranslated region. Multiple amino acid sequence comparisons and phylogenetic analyses indicated that Hc-inhba is closely related to mollusks. Real-time quantitative PCR results showed that Hc-inhba was expressed in all six tissues of H. cumingii. Except for the foot, the expression of Hc-inhba in all other tissues was significantly different (P < 0.05). During early gonadal development, the expression of Hc-inhba showed a trend of increasing and then decreasing, and the expression was significantly higher at the age of four to six months than at age of other months (P < 0.05), with the highest expression of Hc-inhba at the age of six months. During female gonadal development, the expression of Hc-inhba was significantly higher in the growth, maturation and discharge phases than in the proliferation and resting phases (P < 0.05), suggesting that Hc-inhba may be involved in oocyte maturation and ovulation. The results of RNA interference showed that both interfering strand 1 and interfering strand 2 had a certain interfering effect, with the highest interfering rate of 42% in females and 92% in males, and the expression of Hc-inhba-related genes showed different degrees of decrease after interference, predicting the regulatory effect of Hc-inhba. The results of dual luciferase assay showed a significant decrease in firefly luciferase activity in HEK293T cells transfected with wild-type vector and miR-9-5p mimic (P < 0.05), while there was no significant difference in the mutant vector group (P > 0.05). After injection of miR-9-5p antagomir, there was a highly significant decrease (P < 0.01) in the expression of miR-9-5p in both females and males, whereas the expression of Hc-inhba increased highly significantly (P < 0.01). The above results suggest that Hc-inhba may play an important role in the gonadal development of the sail mussel, and miR-9-5p possesses the ability to regulate Hc-inhba.