Lv Jing, Zhu Shibing, Chen Huiping, Xu Ying, Su Qingyu, Yu Guofen, Ma Wei
Department of Traditional Chinese Medicine, Zhejiang Chinese Medicine and Western Medicine Integrated Hospital, Hangzhou, China.
Department of Endocrinology, Zhe Jiang Chinese Medicine and Western Medicine Integrated Hospital, Hangzhou, China.
Drug Dev Res. 2022 Apr;83(2):432-446. doi: 10.1002/ddr.21873. Epub 2021 Oct 12.
Paeonol exerted an effect in lung cancer, but the underlying mechanism remained vague. In this research, we assessed the effects of Paeonol and microRNA (miR)-126-5p on the viability, migration, invasion, and epithelial-mesenchymal transition (EMT) of lung cancer cells. Lung cancer cells and BEAS-2B cells were treated with Paeonol, and viability was detected by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay. The migration and invasion of lung cancer cells after treatment with Paeonol at 40 μg/mL or 80 μg/mL were detected by wound healing assay and Transwell assay, respectively. The effects of Paeonol on transforming growth factor-β1 (TGF-β1)-induced EMT and relative expressions of EMT-related proteins were determined using Western blot. The target gene of miR-126-5p and the binding sites between them were predicted by TargetScan, and confirmed using dual-luciferase reporter assay. Relative expressions of miR-126-5p, its target gene and EMT-related proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Rescue assay was performed to analyze the relation between Paeonol and miR-126-5p. Paeonol down-regulated cell viability and inhibited migration, invasion and TGF-β1-induced EMT while up-regulating miR-126-5p expression in lung cancer cells as the dose increased. However, miR-126-5p inhibitor could reverse the effect of Paeonol. ZEB2 was the target gene of miR-126-5p, and silencing ZEB2 expression reversed the effects of miR-126-5p downregulation. Paeonol also regulated the expression of ZEB2 in lung cancer cells, and this regulation depends on the regulation of miR-126-5p. Paeonol inhibits human lung cancer cell viability and metastasis via the miR-126-5p/ZEB2 axis, and could be adopted as a potential agent for lung cancer treatment.
丹皮酚对肺癌有作用,但其潜在机制仍不明确。在本研究中,我们评估了丹皮酚和微小RNA(miR)-126-5p对肺癌细胞活力、迁移、侵袭及上皮-间质转化(EMT)的影响。用丹皮酚处理肺癌细胞和BEAS-2B细胞,通过3-(4,5)-二甲基噻唑-2,5-二苯基四氮唑溴盐(MTT)法检测细胞活力。分别通过划痕实验和Transwell实验检测40μg/mL或80μg/mL丹皮酚处理后肺癌细胞的迁移和侵袭能力。用蛋白质免疫印迹法测定丹皮酚对转化生长因子-β1(TGF-β1)诱导的EMT及EMT相关蛋白相对表达的影响。通过TargetScan预测miR-126-5p的靶基因及其二者之间的结合位点,并采用双荧光素酶报告基因实验进行验证。通过定量实时聚合酶链反应(qRT-PCR)和蛋白质免疫印迹法测定miR-126-5p、其靶基因及EMT相关蛋白的相对表达。进行挽救实验以分析丹皮酚与miR-126-5p之间的关系。随着剂量增加,丹皮酚下调肺癌细胞活力,抑制迁移、侵袭及TGF-β1诱导的EMT,同时上调miR-126-5p表达。然而,miR-126-5p抑制剂可逆转丹皮酚的作用。锌指E盒结合蛋白2(ZEB2)是miR-126-5p的靶基因,沉默ZEB2表达可逆转miR-126-5p下调的作用。丹皮酚还可调节肺癌细胞中ZEB2的表达,且这种调节依赖于miR-126-5p的调节。丹皮酚通过miR-126-5p/ZEB2轴抑制人肺癌细胞活力和转移,可作为肺癌治疗的潜在药物。