Cloning and Characterization of Yak DHODH Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts

Simple SummaryIn recent years, authoritative journals have consistently reported DHODH’s critical role in resistance to ferroptosis, significantly advancing the research on related mechanisms. Nevertheless, the nucleotide sequence of the yakDHODHgene remains unknown, and its involvements in ferroptosis processes in yak cells remain elusive. Therefore, in the present study, we cloned the coding region of the yakDHODHgene, and revealed its high conservation in mammals. Furthermore, by using a bisphenol S (BPS)-induced ferroptosis model, we confirmed the role of theDHODHgene in mitigating ferroptosis in yak skin fibroblasts (YSFs) derived from a three-mouth-old fetus. This study offers theoretical support for further exploring the functions of the yakDHODHgene and partially elucidating the molecular mechanisms underlying ferroptosis.AbstractDihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme ofde novobiosynthesis of pyrimidine. Although the involvement of DHODH in resisting ferroptosis has been successively reported in recent years, which greatly advanced the understanding of the mechanism of programmed cell death (PCD), the genetic sequence of the yakDHODHgene and its roles in ferroptosis are still unknown. For this purpose, we firstly cloned the coding region sequence ofDHODH(1188 bp) from yak liver and conducted a characterization analysis of its predictive protein that consists of 395 amino acids. We found that the coding region of the yakDHODHgene presented high conservation among species. Second, the expression profile of theDHODHgene in various yak tissues was investigated using RT-qPCR. The results demonstrated thatDHODHwas widely expressed in different yak tissues, with particularly high levels in the spleen, heart, and liver. Third, to investigate the involvement ofDHODHin regulating ferroptosis in cells, yak skin fibroblasts (YSFs) were isolated from fetuses. And then, bisphenol S (BPS) was used to induce thein vitroferroptosis model of YSFs. We observed that BPS decreased the cell viability (CCK8) and membrane potential (JC-1) of YSFs in a dose-dependent manner and induced oxidative stress by elevating reactive oxygen species (ROS). Simultaneously, it was evident that BPS effectively augmented the indicators associated with ferroptosis (MDA and BODIPY staining) and reduced GSH levels. Importantly, the co-administration of Ferrostatin-1 (Fer), a potent inhibitor of ferroptosis, significantly alleviated the aforementioned markers, thereby confirming the successful induction of ferroptosis in YSFs by BPS. Finally, overexpression plasmids and siRNAs of the yakDHODHgene were designed and transfected respectively into BPS-cultured YSFs to modulateDHODHexpression. The findings revealed thatDHODHoverexpression alleviated the occurrence of BPS-induced ferroptosis, while interference ofDHODHintensified the ferroptosis process in YSFs. In summary, we successfully cloned the coding region of the yakDHODHgene, demonstrating its remarkable conservation across species. Moreover, using BPS-induced ferroptosis in YSFs as the model, the study confirmed the role of theDHODHgene in resisting ferroptosis in yaks. These results offer valuable theoretical foundations for future investigations into the functionality of the yakDHODHgene and the underlying mechanisms of ferroptosis in this species.Keywords:yak;DHODHgene;fetal fibroblasts;ferroptosis;Bisphenol S