Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts
Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of <i>de novo</i> biosynthesis 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...
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MDPI AG
2023-12-01
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| author | Hongmei Xu Yueyue Li Qiao Li Zifeng Ma Shi Yin Honghong He Yan Xiong Xianrong Xiong Daoliang Lan Jian Li Wei Fu |
| author_facet | Hongmei Xu Yueyue Li Qiao Li Zifeng Ma Shi Yin Honghong He Yan Xiong Xianrong Xiong Daoliang Lan Jian Li Wei Fu |
| author_sort | Hongmei Xu |
| collection | DOAJ |
| description | Dihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of <i>de novo</i> biosynthesis 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 yak <i>DHODH</i> gene and its roles in ferroptosis are still unknown. For this purpose, we firstly cloned the coding region sequence of <i>DHODH</i> (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 yak <i>DHODH</i> gene presented high conservation among species. Second, the expression profile of the <i>DHODH</i> gene in various yak tissues was investigated using RT-qPCR. The results demonstrated that <i>DHODH</i> was widely expressed in different yak tissues, with particularly high levels in the spleen, heart, and liver. Third, to investigate the involvement of <i>DHODH</i> in regulating ferroptosis in cells, yak skin fibroblasts (YSFs) were isolated from fetuses. And then, bisphenol S (BPS) was used to induce the <i>in vitro</i> ferroptosis 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 yak <i>DHODH</i> gene were designed and transfected respectively into BPS-cultured YSFs to modulate <i>DHODH</i> expression. The findings revealed that <i>DHODH</i> overexpression alleviated the occurrence of BPS-induced ferroptosis, while interference of <i>DHODH</i> intensified the ferroptosis process in YSFs. In summary, we successfully cloned the coding region of the yak <i>DHODH</i> gene, demonstrating its remarkable conservation across species. Moreover, using BPS-induced ferroptosis in YSFs as the model, the study confirmed the role of the <i>DHODH</i> gene in resisting ferroptosis in yaks. These results offer valuable theoretical foundations for future investigations into the functionality of the yak <i>DHODH</i> gene and the underlying mechanisms of ferroptosis in this species. |
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| spelling | doaj.art-833df95838e34f5c814b0f14aa8d2af52023-12-22T13:47:32ZengMDPI AGAnimals2076-26152023-12-011324383210.3390/ani13243832Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal FibroblastsHongmei Xu0Yueyue Li1Qiao Li2Zifeng Ma3Shi Yin4Honghong He5Yan Xiong6Xianrong Xiong7Daoliang Lan8Jian Li9Wei Fu10Key Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaKey Laboratory of Animal Science of National Ethnic Affairs Commission of China, Southwest Minzu University, Chengdu 610041, ChinaDihydroorotate dehydrogenase (DHODH) is a rate-limiting enzyme of <i>de novo</i> biosynthesis 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 yak <i>DHODH</i> gene and its roles in ferroptosis are still unknown. For this purpose, we firstly cloned the coding region sequence of <i>DHODH</i> (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 yak <i>DHODH</i> gene presented high conservation among species. Second, the expression profile of the <i>DHODH</i> gene in various yak tissues was investigated using RT-qPCR. The results demonstrated that <i>DHODH</i> was widely expressed in different yak tissues, with particularly high levels in the spleen, heart, and liver. Third, to investigate the involvement of <i>DHODH</i> in regulating ferroptosis in cells, yak skin fibroblasts (YSFs) were isolated from fetuses. And then, bisphenol S (BPS) was used to induce the <i>in vitro</i> ferroptosis 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 yak <i>DHODH</i> gene were designed and transfected respectively into BPS-cultured YSFs to modulate <i>DHODH</i> expression. The findings revealed that <i>DHODH</i> overexpression alleviated the occurrence of BPS-induced ferroptosis, while interference of <i>DHODH</i> intensified the ferroptosis process in YSFs. In summary, we successfully cloned the coding region of the yak <i>DHODH</i> gene, demonstrating its remarkable conservation across species. Moreover, using BPS-induced ferroptosis in YSFs as the model, the study confirmed the role of the <i>DHODH</i> gene in resisting ferroptosis in yaks. These results offer valuable theoretical foundations for future investigations into the functionality of the yak <i>DHODH</i> gene and the underlying mechanisms of ferroptosis in this species.https://www.mdpi.com/2076-2615/13/24/3832yak<i>DHODH</i> genefetal fibroblastsferroptosisBisphenol S |
| spellingShingle | Hongmei Xu Yueyue Li Qiao Li Zifeng Ma Shi Yin Honghong He Yan Xiong Xianrong Xiong Daoliang Lan Jian Li Wei Fu Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts Animals yak <i>DHODH</i> gene fetal fibroblasts ferroptosis Bisphenol S |
| title | Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts |
| title_full | Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts |
| title_fullStr | Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts |
| title_full_unstemmed | Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts |
| title_short | Cloning and Characterization of Yak <i>DHODH</i> Gene and Its Functional Studies in a Bisphenol S-Induced Ferroptosis Model of Fetal Fibroblasts |
| title_sort | cloning and characterization of yak i dhodh i gene and its functional studies in a bisphenol s induced ferroptosis model of fetal fibroblasts |
| topic | yak <i>DHODH</i> gene fetal fibroblasts ferroptosis Bisphenol S |
| url | https://www.mdpi.com/2076-2615/13/24/3832 |
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