Abstract:Pleomorphic adenoma gene 1 (PLAG1) plays a crucial role in regulating the growth and development of livestock and poultry. Our previous study identified that PLAG1 was associated with backfat thickness (BF) in pigs by genome-wide association study (GWAS), suggesting a potential regulatory effect on adipocytes. To further investigate the relationship between PLAG1 gene and adipocytes, 3T3-L1 cells was used as a research model and PLAG1 conservation was analyzed. The effects of PLAG1 gene overexpression on cell proliferation and differentiation were examined using qRT-PCR, CCK-8 assays, flow cytometry, and Oil Red O staining. Results revealed high conservation of the PLAG1 gene, with sequence similarity among different species ranging from 80% to 92%. qRT-PCR analysis showed that, compared with the control group, the expression of proliferation-related genes in 3T3-L1 cells was extremely significantly increased following PLAG1 overexpression (P<0.01). CCK-8 assays showed that PLAG1 overexpression extremely significantly enhanced cell viability (P<0.01). Flow cytometry demonstrated a significant increase in the number of cells in the S phase following PLAG1 overexpression (P<0.05). Furthermore, Oil Red O staining indicated that the expression of adipogenic differentiation-related genes was significantly decreased after PLAG1 overexpression (P<0.05), accompanied by a extremely significant reduction in lipid droplet formation (P<0.01). In summary, PLAG1 overexpression promoted the proliferation of 3T3-L1 cells while inhibiting their differentiation, indicating that PLAG1 gene might play a regulatory role in the proliferation and differentiation of adipocytes. This study provides a theoretical foundation for further investigation of the molecular mechanisms through which the PLAG1 gene regulates fat deposition in pigs.
吴姿仪, 白利瑶, 吴嘉浩, 窦腾飞, 李新建, 韩雪蕾, 乔瑞敏, 王克君, 杨峰, 李秀领. 猪PLAG1基因过表达对3T3-L1前脂肪细胞增殖分化的影响[J]. 农业生物技术学报, 2025, 33(4): 782-791.
WU Zi-Yi, BAI Li-Yao, WU Jia-Hao, DOU Teng-Fei, LI Xin-Jian, HAN Xue-Lei, QIAO Rui-Min, WANG Ke-Jun, YANG Feng, LI Xiu-Ling. Effects of Overexpression of Porcine (Sus Scrofa) PLAG1 Gene on Proliferation and Differentiation of 3T3-L1 Preadipocytes. 农业生物技术学报, 2025, 33(4): 782-791.
[1] 李世军. 2020. 牛PLIN1基因转录调控机制及其对前体脂肪细胞增殖、分化和脂类代谢的作用研究[M]. 博士学位论文 , 西北农林科技大学, 导师: 昝林森, pp. 38.(Transcriptional regulation mechanism of bovine PLIN1 gene and its effect onproliferation, differentiation and lipidmetabolism of preadipocytes[M]. Thesis for Ph.D., Northwest A&F University, Suppervisor: Zan L S, pp. 38.) [2] 王洋. 2013. PLAG1在细胞分化及转基因小鼠唾液腺肿瘤中的作用[D]. 博士学位论文, 上海交通大学, 导师: 张陈平, pp. 38. (Wang Y. 2013. A studay of PLAG1 on regulation of cellular differentiation and tumor formation in transgenic mice[D]. Thesis for Ph.D., Shanghai Jiao Tong University, Suppervisor: Zhang C P, pp. 38.) [3] 赵蕾, 郑美丽, 杨梅, 等. 2019. 3T3-L1前体脂肪细胞诱导分化方法优化的初步探讨[J]. 首都医科大学学报, 40: 226-31.(Zhao L, Zheng M L, Yang M, et al.2019. Optimization of induction and differentiation of 3T3-L1 preadipocyte[J]. Journal of Capital Medical University, 40: 226-231.) [4] 朱美琪. 2023. ACSS3基因对3T3-L1前体脂肪细胞增殖分化的作用机制研究[D]. 硕士学位论文, 山东农业大学, 导师: 曾勇庆, pp. 69. (Zhu M Q. 2023. Mechanism of ACSS3 gene on proliferation and differentiation of 3T3-L1 preadipocytes[D]. Thesis for M.S., Shandong Agricultural University, Suppervisor: Zeng Y Q, pp. 69.) [5] Abdalla B A, Li Z, Ouyang H, et al.2018. A novel Dnmt3a1 transcript inhibits adipogenesis[J]. Frontiers in Physiology, 9: 1270. [6] Ambele M A, Dhanraj P, Giles R, et al.2020. Adipogenesis: A complex interplay of multiple molecular determinants and pathways[J]. International Journal of Molecular Sciences, 21(12): 4283. [7] Beezhold K, Klei L R, Barchowsky A.2017. Regulation of cyclin D1 by arsenic and microRNA inhibits adipogenesis[J]. Toxicology Letters, 265: 147-155. [8] Cheng P Q, Liu Y J, Zhang S A, et al.2022. RNA-seq profiling of circular RNAs in human colorectal cancer 5-fluorouracil resistance and potential biomarkers[J]. World Journal of Gastrointestinal Oncology, 14: 678-689. [9] Corvò R, Giaretti W, Sanguineti G, et al.1995. In vivo cell kinetics in head and neck squamous cell carcinomas predicts local control and helps guide radiotherapy regimen[J]. Journal of Clinical Oncology, 13: 1843-1850. [10] Dadousis C, Somavilla A, Ilska J J, et al.2021. A genome-wide association analysis for body weight at 35 days measured on 137,343 broiler chickens[J]. Genetics Selection Evolution, 53: 70. [11] de Melo K M, de Oliveira F T B, Costa Silva R A, et al.2019. α, β-Amyrin, a pentacyclic triterpenoid from Protium heptaphyllum suppresses adipocyte differentiation accompanied by down regulation of PPARγ and C/EBPα in 3T3-L1 cells[J]. Biomedicine & Pharmacotherapy, 109: 1860-1866. [12] Domm J M, Wootton S K, Medin J A, et al.2021. Gene therapy for Fabry disease: Progress, challenges, and outlooks on gene-editing[J]. Molecular Genetics and Metabolism, 134: 117-131. [13] Gregoire F M, Smas C M, Sul H S.1998. Understanding adipocyte differentiation[J]. Physiological Reviews, 78: 783-809. [14] Gudbjartsson D F, Walters G B, Thorleifsson G, et al.2008. Many sequence variants affecting diversity of adult human height[J]. Nature Genetics, 40: 609-615. [15] Hoshiba H, Setoguchi K, Watanabe T, et al.2013. Comparison of the effects explained by variations in the bovine PLAG1 and NCAPG genes on daily body weight gain, linear skeletal measurements and carcass traits in Japanese Black steers from a progeny testing program[J]. Animal Science Journal, 84: 529-534. [16] Jang H J, Kim H Y, Lyu J H, et al.2024. Bee (Apis mellifera L. 1758) wax restores adipogenesis and lipid accumulation of 3T3-L1 cells in cancer-associated cachexia condition[J]. Food Science and Nutrition, 12: 5027-5035. [17] Juma A R, Damdimopoulou P E, Grommen S V, et al.2016. Emerging role of PLAG1 as a regulator of growth and reproduction[J]. Journal of Endocrinology, 228: R45-56. [18] Juma A R, Grommen S V H, O'bryan M K, et al.2017. PLAG1 deficiency impairs spermatogenesis and sperm motility in mice[J]. Scientific Reports, 7: 5317. [19] Karim L, Takeda H, Lin L, et al.2011. Variants modulating the expression of a chromosome domain encompassing PLAG1 influence bovine stature[J]. Nature Genetics, 43: 405-413. [20] Landrette S F, Kuo Y H, Hensen K, et al.2005. Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11[J]. Blood, 105: 2900-2907. [21] Lee J E, Schmidt H, Lai B, et al.2019. Transcriptional and epigenomic regulation of adipogenesis[J]. Molecular and Cellular Biology, 39(11): e00601-18. [22] Lim S, Kaldis P.2013. Cdks, cyclins and CKIs: Roles beyond cell cycle regulation[J]. Development, 140: 3079-3093. [23] Merkel D E, Mcguire W L.1990. Ploidy, proliferative activity and prognosis. DNA flow cytometry of solid tumors[J]. Cancer, 65: 1194-1205. [24] Pan M, Sun Q, Li C, et al.2022a. Hoxa5 inhibits adipocyte proliferation through transcriptional regulation of Ccne1 and blocking JAK2/STAT3 signaling pathway in mice[J]. International Journal of Biochemistry & Cell Biology, 100: 325-337. [25] Pan Y, Wang M, Wu H, et al.2022b. Indel mutations of sheep PLAG1 gene and their associations with growth traits[J]. Animal Biotechnology, 33: 1459-1465. [26] Qiao R, Gao J, Zhang Z, et al.2015. Genome-wide association analyses reveal significant loci and strong candidate genes for growth and fatness traits in two pig populations[J]. Genetics Selection Evolution, 47: 17. [27] Rajender S, Avery K, Agarwal A.2011. Epigenetics, spermatogenesis and male infertility[J]. Mutation Research-reviews In Mutation Research, 727: 62-71. [28] Rubin C J, Megens H J, Martinez Barrio A, et al.2012. Strong signatures of selection in the domestic pig genome[J]. Proceedings of the National Academy of Sciences of the USA, 109: 19529-19536. [29] Song Y, Xu L, Chen Y, et al.2016. Genome-wide association study reveals the PLAG1 gene for knuckle, biceps and shank weight in Simmental beef cattle[J]. Public Library of Science ONE, 11: e0168316. [30] Takasuga A.2016. PLAG1 and NCAPG-LCORL in livestock[J]. Journal of Animal Science, 87: 159-167. [31] Tamura K, Stecher G, Kumar S.2021. MEGA11: Molecular evolutionary genetics analysis version 11[J]. Molecular Biology and Evolution, 38: 3022-3027. [32] Tang Q Q, Otto T C, Lane M D2003. CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis[J]. Proceedings of the National Academy of Sciences of the USA, 100: 850-855. [33] Van Dyck F, Declercq J, Braem C V, et al.2007. PLAG1, the prototype of the PLAG gene family: Versatility in tumour development (review)[J]. International Journal of Oncology, 30: 765-774. [34] Wang X, Li X, Wang Y, et al.2024. Nkx1.2 deletion decreases fat production in zebrafish[J]. Obesity (Silver Spring), 32: 1315-1328. [35] Wong J, Juma A R, Tran S C, et al.2020. Deficiency of the transcription factor PLAG1 results in aberrant coiling and morphology of the epididymis[J]. Asian Journal of Andrology, 22: 342-347. [36] Wu Z, Dou T, Bai L, et al.2024. Genomic prediction and genome-wide association studies for additive and dominance effects for body composition traits using 50 K and imputed high-density SNP genotypes in Yunong-black pigs[J]. Journal of Animal Breeding and Genetics, 141: 124-137. [37] Xiao C, Jin H G, Zhang L C, et al.2021. Effects of SPARCL1 on the proliferation and differentiation of sheep preadipocytes[J]. Adipocyte, 10: 658-669. [38] Xu P, Li D, Wu Z, et al.2022. An imputation-based genome-wide association study for growth and fatness traits in Sujiang pigs[J]. Animal, 16: 100591. [39] Xu P, Ni L, Tao Y, et al.2020. Genome-wide association study for growth and fatness traits in Chinese Sujiang pigs[J]. Animal Genetics, 51: 314-318. [40] Xu W, He H, Zheng L, et al.2018. Detection of 19-bp deletion within PLAG1 gene and its effect on growth traits in cattle[J]. Gene, 675: 144-149. [41] Zhou Z, Huang B, Lai Z, et al.2019. The distribution characteristics of a 19-bp indel of the PLAG1 gene in Chinese cattle[J]. Animals (Basel), 9(12): 1082.