LncRNA HAND2OT encoding micropeptide regulates the estradiol secretion, proliferation and apoptosis of ovarian follicular theca cells in chickens

Long non-coding RNAs (lncRNAs) are critical regulators of ovarian follicular development. In chicken ovarian follicles, theca cells are primarily responsible for androgen and estradiol biosynthesis and play an important role in chicken follicular development, however, the function of lncRNAs in chicken theca cells remains unknown. This study characterized a novel lncRNA HAND2OT (heart and neural crest derivatives expressed 2 overlapping transcript) and elucidated its role in chicken theca cells. With a full length of 950 nt, HAND2OT was localized predominantly in the cytoplasm. The expression of HAND2OT was highest in large white follicles (LW), increased progressively from F6 to F1 follicles, declined in granulosa cells but increased in theca cells following follicle selection. HAND2OT expression was upregulated by estradiol, while downregulated by progesterone and follicle-stimulating hormone (FSH). Functionally, HAND2OT suppressed estradiol secretion, inhibited theca cell proliferation by inducing cell cycle arrest at the G0/G1 phase and promoted apoptosis. The open reading frame 2(ORF2) of HAND2OT encoded a 66-amino-acid peptide (HAND2OT-66aa) exerting no effect on HAND2OT expression. Both the lncRNA HAND2OT lacking the HAND2OT-66aa and HAND2OT-66aa itself exhibited weaker effects on the cell proliferation and apoptosis of theca cells, compared with the full length HAND2OT, suggesting that HAND2OT regulates the survival and function of chicken theca cells via its RNA moiety and the HAND2OT-66aa micropeptide it encodes.