The mechanism of miR-451 on regulating differentiation of 3T3-L1 preadipocytes through AMPK signaling pathway

doi:10.11958/20253387

Abstract

Abstract:

Objective To investigate the role and mechanism of miR-451 in regulating the differentiation of 3T3-L1 preadipocytes through the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. Methods 3T3-L1 cells were randomly divided into the control group, the NC group, the miR-451 mimics group and the miR-451 inhibitor group. The expression of miR-451 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Lipid droplet formation was observed by Oil Red O staining. The targeting relationship between miR-451 and calcium-binding protein 39 (CAB39) was verified by dual-luciferase reporter gene assay. The protein expressions of AMPK and mammalian target of rapamycin (mTOR) were detected by Western blot assay. 3T3-L1 cells with AMPK knockdown were constructed and divided into the NC-N.A group, the miR-451 mimics-N.A group and the miR-451 inhibitor-N.A group. Triglyceride (TG) levels were measured. Results After 7 days of induced differentiation of 3T3-L1 cells into mature adipocytes, the expression of miR-451 was significantly increased (P<0.05). Compared with the control group volume and the NC group, the miR-451 mimics group showed significantly increased miR-451 expression, larger lipid droplets volume and deeper Oil Red O staining (P<0.05). In contrast, the miR-451 inhibitor group exhibited the opposite changes (P<0.05). The dual-luciferase reporter assay confirmed that CAB39 was a direct target gene of miR-451. Western blot results showed that, compared with the control group and the NC group, p-AMPK expression decreased, and p-mTOR expression increased in the miR-451 mimics group (P<0.05), while p-AMPK expression increased and p-mTOR expression decreased in the miR-451 inhibitor group (P<0.05). In AMPK-knockdown 3T3-L1 cells, neither overexpression nor inhibition of miR-451 affected TG production. Conclusion miR-451 can promote the differentiation of 3T3-L1 preadipocytes, and this process is associated with the inhibition of the AMPK signaling pathway by miR-451.

Key words: microRNAs, adipocyte, miR-451, adenylate kinase

CLC Number:

R587.1

Figures/Tables 7

Fig.1 Base complementary pairing of miR-451 and CAB39 mRNA in 3'-UTR region

Fig.2 Expression of miR-451 in the differentiation process of 3T3-L1 preadipocytes

Fig.3 Expression of miR-451 in each group

Fig.4 Effects of miR-451 on differentiation of 3T3-L1 preadipocytes (Oil Red O staining, ×200)

Fig.5 Detection of cell luciferase activity by luciferase reporter gene assay in each group

Fig. 6 Expressions of AMPK signaling pathway proteins in each group

Fig.7 Western blot verification of AMPK knockdown effect in 3T3-L1 preadipocytes

References 20

[1]	Perdomo C M, Cohen R V, Sumithran P, et al. Contemporary medical,device,and surgical therapies for obesity in adults[J]. Lancet, 2023, 401(10382):1116-1130. doi:10.1016/S0140-6736(22)02403-5.
[2]	国家卫生健康委办公厅. 体重管理指导原则(2024年版)[EB/OL].(2024-12-30)[2025-11-03]. https://www.nhc.gov.cn/ylyjs/zcwj/202412/75cb79c171c94def9e768193e65484f7/files/1736390749000_59785.pdf.
	National Health Commission of the People's Republic of China General Office. Guidelines for Weight Management (2024 Edition)[EB/OL]. (2024-12-30)[2025-11-03]. https://www.nhc.gov.cn/ylyjs/zcwj/202412/75cb79c171c94def9e768193e65484f7/files/1736390749000_59785.pdf.
[3]	Sumathipala M, Weiss S T. Predicting mirna-based disease-disease relationships through network diffusion on multi-omics biological data[J]. Sci Rep, 2020, 10(1):8705. doi:10.1038/s41598-020-65633-6.
[4]	孟超群, 李成萍, 赵薇, 等. miR-137-3p靶向MAX基因对前脂肪细胞3T3-L1成脂分化的影响[J]. 中国畜牧兽医, 2023, 50(5):1928-1937.
	Meng C Q, Li C P, Zhao W, et al. MiR-137-3p targets MAX gene to regulate adipogenic differentiation of 3T3-L1 preadipocytes[J]. Chin J Anim Husb Vet Med, 2023, 50(5):1928-1937. doi:10.16431/j.cnki.1671-7236.2023.05.020.
[5]	张寒月, 赵丹, 梁煜, 等. miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2023, 54(8):3262-3274.
	Zhang H Y, Zhao D, Liang Y, et al. MiR-150 targets AOC3 to regulate the differentiation of sheep preadipocytes[J]. Chin J Anim Vet Sci, 2023, 54(8):3262-3274. doi:10.11843/j.issn.0366-6964.2023.08.013.
[6]	Nan Y, Guo L, Zhen Y, et al. MiRNA-451 regulates the NF-κB Signaling pathway by targeting Ikkβ to inhibit glioma cell growth[J]. Cell Cycle, 2021, 20(19):1967-1977. doi:10.1080/15384101.2021.1969496.
[7]	Deng H Y, He Z Y, Dong Z C, et al. MicroRNA-451a attenuates angiotensin II-induced cardiac fibrosis and inflammation by directly targeting T-box1[J]. J Physiol Biochem, 2022, 78(1):257-269. doi:10.1007/s13105-021-00861-6.
[8]	姚俊鹏, 周思远, 鄢香芸, 等. 基于mTOR/P70S6K信号通路探讨电针对肥胖大鼠脂肪组织自噬的影响[J]. 中华中医药杂志, 2022, 37(5):2633-2638.
	Yao J P, Zhou S Y, Yan X Y, et al. Effect of electroacupuncture on autophagy in adipose tissue of obese rats based on the mTOR/P70S6K signaling pathway[J]. China J Tradit Chin Med Pharm, 2022, 37(5):2633-2638.
[9]	杨琼, 辜浩, 堵晶晶, 等. microRNA-425-5p对3T3-L1前体脂肪细胞增殖、分化的影响[J]. 中国畜牧兽医, 2019, 46(2):354-364.
	Yang Q, Gu H, Du J J, et al. Effect of microRNA-425-5p on proliferation and differentiation of 3T3-L1 preadipocytes[J]. Chin J Anim Husb Vet Med, 2019, 46(2):354-364. doi:10.16431/j.cnki.1671-7236.2019.02.004.
[10]	Zhao K, Wang L, Li T, et al. The role of miR-451 in the switching between proliferation and migration in malignant glioma cells:AMPK signaling,mTOR modulation and Rac1 activation required[J]. Int J Oncol, 2017, 50(6):1989-1999. doi:10.3892/ijo.2017.3973.
[11]	Guo R, Gu J, Zhang Z, et al. MiR-451 promotes cell proliferation and metastasis in pancreatic cancer through targeting CAB39[J]. Biomed Res Int, 2017, 2017:2381482. doi:10.1155/2017/2381482.
[12]	倪文婧, 耿楠, 白雪, 等. 《2024年欧洲肝病学会/欧洲糖尿病学会/欧洲肥胖症学会临床实践指南:代谢相关脂肪性肝病的管理》摘译[J]. 临床肝胆病杂志, 2024, 40(8):1567-1574.
	Ni W J, Geng N, Bai X, et al. Abstract translation of 2024 EASL/EASD/EASO clinical practice guidelines for the management of metabolic-associated fatty liver disease[J]. J Clin Hepatol, 2024, 40(8):1567-1574. doi:10.12449/JCH240810.
[13]	Aron-Wisnewsky J, Warmbrunn M V, Nieuwdorp M, et al. Metabolism and metabolic disorders and the microbiome:the intestinal microbiota associated with obesity,lipid metabolism,and metabolic health-pathophysiology and therapeutic strategies[J]. Gastroenterology, 2021, 160(2):573-599. doi:10.1053/j.gastro.2020.10.057.
[14]	周颖, 滕井泉, 田乐乐, 等. 丹参及有效化学成分干预肥胖机制的研究进展[J]. 中药材, 2023, 46(11):2890-2894.
	Zhou Y, Teng J Q, Tian L L, et al. Research progress on the mechanism of Salvia miltiorrhiza and its active chemical components in intervening obesity[J]. J Chin Med Mater, 2023, 46(11):2890-2894. doi:10.13863/j.issn1001-4454.2023.11.044.
[15]	Fang X, Shen F, Lechauve C, et al. miR-144/451 represses the LKB1/AMPK/mTOR pathway to promote red cell precursor survival during recovery from acute anemia[J]. Haematologica, 2018, 103(3):406-416. doi:10.3324/haematol.2017.177394.
[16]	Wei H, Li J, Li Y, et al. MicroRNA-451 inhibits inflammation and proliferation of glomerular mesangial cells through down-regulating PSMD11 and NF-κB p65[J]. Biosci Rep, 2019, 39(10):BSR20191455. doi:10.1042/BSR20191455.
[17]	Gan M, Shen L, Fan Y, et al. Ssc-miR-451 regulates porcine primary adipocyte differentiation by targeting ACACA[J]. Animals (Basel), 2020, 10(10):1891. doi:10.3390/ani10101891.
[18]	刘悦, 余松, 韩永霞, 等. 紫色悬钩子提取物对3T3-L1前脂肪细胞分化的影响[J]. 中国药理学通报, 2023, 39(6):1090-1096.
	Liu Y, Yu S, Han Y X, et al. Effect of Rubus irenaeus Focke extract on differentiation of 3T3-L1 preadipocytes[J]. Chin Pharmacol Bull, 2023, 39(6):1090-1096. doi:10.12360/CPB202201065.
[19]	赵彩霞, 刘建芳, 刘宏, 等. 雷公藤甲素对心力衰竭大鼠心肌损伤及AMPK/mTOR通路的影响[J]. 中南医学科学杂志, 2025, 53(1):23-27,69.
	Zhao C X, Liu J F, Liu H, et al. Effect of triptolide on myocardial injury and AMPK/mTOR pathway in rats with heart failure[J]. J South-Centr Univ Natl Sci Technol (Med Sci), 2025, 53(1):23-27,69. doi:10.15972/j.cnki.43-1509/r.2025.01.005.
[20]	Shi G, Li D, Zhang D, et al. IRF-8/miR-451a regulates M-MDSC differentiation via the AMPK/mTOR signal pathway during lupus development[J]. Cell Death Discov, 2021, 7(1):179. doi:10.1038/s41420-021-00568-z.