Effect of Circadian Clock Gene BMAL1 Overexpression on Apoptosis of Goat (Capra hircus) Endometrial Epithelial Cells
LI Chao*, XU Hao-Dong*, GUO Yi-Ying, LIU Wei, WANG Yi-Qun, XIAO Yao-Yao, YANG Wang-Hao, JIN Ya-Ping**, CHEN Hua-Tao**
College of Veterinary Medicine/Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling 712100, China
Abstract：As an endogenous timekeeping system, the circadian clock system plays an important role in regulating behavioral activities and physiological functions in mammals. To investigate the role of the core clock gene brain and muscle arnt-like protein 1 (BMAL1) in apoptosis of goat endometrial epithelial cells (GEECs), the CDS of BMAL1 gene was cloned from 24-month-old goat (Capra hircus) uterine tissues, and the physicochemical properties of the gene and its coding protein were analyzed and predicted using the bioinformatics software; the expression patterns of BMAL1 were detected using qPCR in different tissues of goats as well as GEECs after synchronization. The effect of BMAL1 gene overexpression on GEECs apoptosis was detected by flow cytometry, qPCR, and Western blot (WB). The results showed that the expression of BMAL1 gene showed significant diurnal differences in various tissues and at different time points. The total length of CDS fragment of goat BMAL1 gene was 1 881 bp, which was highly conserved in mammals. And protein encoded by BMAL1 could participate in multiple physiological processes by interacting with proteins such as circadian locomotor output cycles kaput (CLOCK), nuclear receptor subfamily 1, group D, member 1 (NR1D1), and casein kinase 1δ (CSNK1D). Flow cytometry results showed that BMAL1 overexpression significantly reduced the apoptosis rate in GEECs (P<0.05). qPCR and Western blot results showed that BMAL1 overexpression led to a significant decrease in the expression levels of pro-apoptotic genes BCL2-associated X protein (BAX) and tumor suppressor gene P53 in both mRNA and protein, and the expression of apoptosis suppressor gene B cell lymphoma-2 (BCL-2) significantly elevated (P<0.05). This study provides a preliminary basis for analyzing the molecular regulatory mechanism of the ruminant circadian clock system and further exploring the biological functions of the goat BMAL1 gene.
[1] 丁赫, 郭乐薇, 徐高青, 等. 2019. BMAL1对小鼠睾丸间质细胞凋亡的影响[J]. 中国畜牧杂志, 55(06): 63-67. (Ding H, Guo L W, Xu G Q, et al.2019. Effect of BMAL1 on apoptosis in mouse leydig cells[J]. Chinese Journal of Animal Science, 55(06): 63-67.) [2] 付世新, 邢菲菲, 罗胜缤, 等. 2021. NEFA对奶牛子宫内膜上皮细胞凋亡因子基因表达的影响[J]. 黑龙江八一农垦大学学报, 33(04): 40-44. (Fu S X, Xing F F, Luo S B, et al.2021. Effect of NEFA on gene expression of apoptotic factors in endometrial epithelial cells of dairy cows[J]. Journal of Heilongjiang Bayi Agricultural University, 33(04): 40-44.) [3] 牟佼, 戴进前, 刘明莉, 等. 2018. 敲除BMAL1基因促进HL-60细胞凋亡并抑制增殖[J]. 中国实验血液学杂志, 26(04): 1027-1032. (Mu J, Dai J Q, Liu M L, et al.2018. Knockout of BMAL1 gene induces apoptosis of HL-60 cells and inhibits its proliferation[J]. Journal of Experimental Hematology, 26(04): 1027-1032.) [4] 牛宏宇, 张瑞雪, 齐茂振, 等. 2022. 山羊CREBZF蛋白两种亚型过表达载体的构建及其对子宫内膜上皮细胞凋亡的影响[J]. 畜牧兽医学报, 53(01): 132-140. (Niu H Y, Zhang R X, Qi M Z, et al.2022. Construction and effect of two CREBZF isoforms overexpression vectors on goat endometrial epithelial cells apoptosis[J]. Acta Veterinaria et Zootechnica Sinica, 53(01): 132-140.) [5] 王逸群, 高登科, 赵泓淙, 等. 2022. 山羊NR1D1基因真核表达载体的构建及生物信息学分析[J]. 中国畜牧杂志, 58(10): 215-222. (Wang Y Q, Gao D K, Zhao H C, et al.2022. Construction of a eukaryotic expression vector of goat NR1D1 gene and its bioinformatics analysis[J]. Chinese Journal of Animal Science, 58(10): 215-222.) [6] 王逸群, 刘祖培, 李雅婷, 等. 2023. 奶牛NR1D1基因的真核表达载体构建、表达谱及其在卵巢组织的定位[J]. 畜牧兽医学报, 54(01): 133-145. (Wang Y Q, Liu Z P, Li Y T, et al.2023. The dairy cow NR1D1 gene's eukaryotic expression vector construction, expression profile and its ovarian localization[J]. Acta Veterinaria et Zootechnica Sinica, 54(01): 133-145.) [7] 吴庆侠, 王爱华, 司永嘉, 等. 2010. 山羊子宫内膜上皮细胞转染pCI-neo-hTERT质粒后的永生化[J]. 中国兽医学报, 30(02): 228-232. (Wu Q X, Wang A H, Si Y J, et al.2010. Immortalization of goat endometrial epithelial cells by telomerase reverse transcriptase transfection[J]. Chinese Journal of Veterinary Science, 30(02): 228-232.) [8] 徐皓东, 王逸群, 刘祖培, 等. 2023. 奶牛生物钟基因BMAL1真核表达载体的构建及生物信息学分析[J]. 中国畜牧杂志, 59(04): 161-167. (Xu H D, Wang Y Q, Liu Z P, et al.2023. Construction of a eukaryotic expression vector and bioinformatics analysis of BMAL1 gene in dairy cow[J]. Chinese Journal of Animal Science, 59(04): 161-167.) [9] 尹雪婷. 2022. ATG7在脂多糖诱导的山羊子宫内膜上皮细胞凋亡中的作用研究[D]. 硕士学位论文, 华中农业大学, 导师: 陈建国. pp. 16-17. (Yin X T.2022. Studies of lipopolysaccharide-induced apoptosis in goat endometrial epithelial cells[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Chen J G, pp. 16-17.) [10] Alvarez J D, Hansen A, Ord T, et al.2008. The circadian clock protein BMAL1 is necessary for fertility and proper testosterone production in mice[J]. Journal of Biological Rhythms, 23(1): 26-36. [11] Early J O, Curtis A M.2016. Immunometabolism: Is it under the eye of the clock?[J]. Seminars in Immunology, 28(5): 478-490. [12] Galan A, O'Connor J E, Valbuena D, et al.2000. The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion[J]. Biology of Reproduction, 63(2): 430-439. [13] Gao D K, Zhao H C, Dong H, et al.2022. Transcriptional feedback loops in the caprine circadian clock system[J]. Frontiers in Veterinary Science, 9: 814562. [14] Harada T, Kaponis A, Iwabe T, et al.2004. Apoptosis in human endometrium and endometriosis[J]. Human Reproduction Update, 10(1): 29-38. [15] Hemmeryckx B, Himmelreich U, Hoylaerts M F, et al.2011. Impact of clock gene Bmal1 deficiency on nutritionally induced obesity in mice[J]. Obesity, 19(3): 659-661. [16] He Q Y, Jin F, Li Y Y, et al.2018. Prognostic significance of downregulated BMAL1 and upregulated Ki-67 proteins in nasopharyngeal carcinoma[J]. Chronobiology International, 35(3): 348-357. [17] Kwon I, Lee J, Chang S H, et al.2006. BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer[J]. Molecular and Cellular Biology, 26(19): 7318-7330. [18] Marcheva B, Ramsey K M, Buhr E D, et al.2010. Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes[J]. Nature, 466(7306): 627-631. [19] Moore L, Leongamornlert D, Coorens T H H, et al.2020. The mutational landscape of normal human endometrial epithelium[J]. Nature, 580(7805): 640-646. [20] Partch C L, Green C B, Takahashi J S.2014. Molecular architecture of the mammalian circadian clock[J]. Trends in Cell Biology, 24(2): 90-99. [21] Qu M, Zhang G, Qu H, et al.2023. Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle[J]. Proceedings of the National Academy of Sciences of the USA, 120(2): e2214829120. [22] Ratajczak C K, Boehle K L, Muglia L J.2009. Impaired steroidogenesis and implantation failure in Bmal1-/- mice[J]. Endocrinology, 150(4): 1879-1885. [23] Ratajczak C K, Herzog E D, Muglia L J.2010. Clock gene expression in gravid uterus and extra-embryonic tissues during late gestation in the mouse[J]. Reproduction Fertility and Development, 22(5): 743-750. [24] Tasaki H, Zhao L J, Isayama K, et al.2013. Profiling of circadian genes expressed in the uterus endometrial stromal cells of pregnant rats as revealed by DNA microarray coupled with RNA interference[J]. Frontiers in Endocrinology, 4: 82. [25] Wang A Q, Bei Z, Yue Z, et al.2022. Melatonin reduces apoptosis resistance of hepatocellular carcinoma cells by inhibiting BMAL1[J]. Integrative Cancer Therapies, 21: 1-15. [26] Wang W, Yin L, Bai L, et al.2017. Bmal1 interference impairs hormone synthesis and promotes apoptosis in porcine granulosa cells[J]. Theriogenology, 99: 63-68. [27] Wei H, Qu L, Dai S, et al.2021. Structural insight into the molecular mechanism of p53-mediated mitochondrial apoptosis[J]. Nature Communications, 12(1): 2280. [28] Xiao Y Y, Zhao L J, Li W D, et al.2021. Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells[J]. Journal of Cellular Physiology, 236(9):6706-6725.
