Yang Zhi, Wu Weigang, Ou Pengcheng, Wu Minna, Zeng Furong, Zhou Boping, Wu Shipin
Department of Infectious Disease, Shenzhen People's Hospital, The Second Affiliated Clinical Medical College of Jinan University, No.1017, North Dongmen Road, Luohu District, Shenzhen, 518000, Guangdong, China.
Department of Infectious Disease, Shenzhen People's Hospital Longhua Branch, No.101, East Longguan Road, Longhua Street, Longhua District, Shenzhen, 518000, Guangdong, China.
Mol Cell Biochem. 2021 Feb;476(2):1257-1267. doi: 10.1007/s11010-020-03988-0. Epub 2020 Nov 28.
MiR-122-5p serves as a novel biomarker for drug-induced liver injury (DILI), but its function in DILI remains unclear. The present study, therefore, explored the function and potential mechanism of miR-122-5p in DILI. Sprague-Dawley (SD) rats were treated with miR-122-5p antagomir, and then DILI was induced in the rats by acetaminophen (APAP). To determine the effect of miR-122-5p on DILI in vivo, liver injury was examined by HE staining and TUNEL assays, and the levels of serum ALT and AST were determined using an automated clinical chemistry analyzer. To further reveal the mechanism of miR-122-5p in DILI, THLE-2 (normal liver cell line) cells were transfected with miR-122-5p mimic and inhibitor, NDRG3, and siNDRG3, and then injured by APAP. The relationship between miR-122-5p and NDRG3 was determined by TargetScan, luciferase reporter assay, and Western blot. The viability and apoptosis of THLE-2 cells were detected by CCK-8 and flow cytometry, respectively. The levels of mRNA and protein in vivo and in vitro were measured by qRT-PCR and Western blot, respectively. APAP induced liver injury and increased the levels of ALT, AST, and miR-122-5p in DILI rats. However, these effects of APAP were attenuated by miR-122-5p antagomir. MiR-122-5p negatively regulated NDRG3 expression. APAP decreased cell viability, apoptosis resistance, and Bcl-w and Bcl-2 levels whereas increased Bax level in THLE-2 cells. However, these effects of APAP on THLE-2 cells were promoted by miR-122-5p up-regulation but inhibited by miR-122-5p knockdown. MiR-122-5p knockdown protects against APAP-mediated liver injury through up-regulating NDRG3.
微小RNA-122-5p（miR-122-5p）作为药物性肝损伤（DILI）的一种新型生物标志物，但其在DILI中的功能仍不清楚。因此，本研究探讨了miR-122-5p在DILI中的功能及潜在机制。将Sprague-Dawley（SD）大鼠用miR-122-5p拮抗剂处理，然后用对乙酰氨基酚（APAP）诱导大鼠发生DILI。为了确定miR-122-5p在体内对DILI的影响，通过苏木精-伊红（HE）染色和末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）试验检测肝损伤情况，并使用自动临床化学分析仪测定血清谷丙转氨酶（ALT）和谷草转氨酶（AST）水平。为了进一步揭示miR-122-5p在DILI中的机制，将miR-122-5p模拟物、抑制剂、N-myc下游调节基因3（NDRG3）和小干扰RNA（siNDRG3）转染到人正常肝细胞系THLE-2细胞中，然后用APAP损伤细胞。通过TargetScan、荧光素酶报告基因试验和蛋白质免疫印迹法确定miR-122-5p与NDRG3之间的关系。分别通过细胞计数试剂盒-8（CCK-8）法和流式细胞术检测THLE-2细胞的活力和凋亡情况。体内和体外的信使核糖核酸（mRNA）和蛋白质水平分别通过实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法进行测定。APAP诱导DILI大鼠肝损伤，并升高其ALT、AST和miR-122-5p水平。然而，miR-122-5p拮抗剂减弱了APAP的这些作用。miR-122-5p负向调节NDRG3的表达。APAP降低了THLE-2细胞的活力、抗凋亡能力以及Bcl-w和Bcl-2水平，而升高了Bax水平。然而，miR-122-5p上调促进了APAP对THLE-2细胞的这些作用，而miR-122-5p敲低则抑制了这些作用。miR-122-5p敲低通过上调NDRG3来保护细胞免受APAP介导的肝损伤。
