天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (2): 125-130.doi: 10.11958/20211879

• 细胞与分子生物学 • 上一篇    下一篇

槲皮素通过miR-101/EZH2轴介导的EMT途径改善NSCLC吉西他滨耐药的机制研究 #br#

钱麒钰,袁改利,马珊珊,罗莉梅,徐玉   

  1. 1郑州大学附属郑州中心医院心肺功能科（邮编450007），2呼吸与危重症医学科
  • 收稿日期:2021-08-16 修回日期:2021-10-14 出版日期:2022-02-15 发布日期:2022-02-15
  • 通讯作者: 钱麒钰 E-mail:wh88hosp@163.com
  • 基金资助:
    2018年河南省科技攻关项目（182102310314）

Study on the mechanism of quercetin improving gemcitabine resistance in NSCLC through the EMT pathway mediated by miR-101/EZH2 axis

QIAN Qiyu, YUAN Gaili, MA Shanshan, LUO Limei, XU Yu #br#   

  1. 1 Department of Cardiopulmonary Function, 2 Department of Respiratory and Critical Care Medicine, Zhengzhou Central
    Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, China
  • Received:2021-08-16 Revised:2021-10-14 Published:2022-02-15 Online:2022-02-15

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摘要： 目的 从微小RNA-101（miR-101）/Zeste基因同源蛋白2（EZH2）轴的角度探究槲皮素逆转非小细胞肺癌 （NSCLC）上皮间质转分化（EMT）及吉西他滨（Gb）耐药性的分子机制。方法 用不同浓度槲皮素培养A549及A549/ Gb，CCK-8法筛选适宜的槲皮素浓度进行后续试验；取A549细胞分为空白组、A549+EMT诱导剂组、A549+槲皮素 组、槲皮素+EMT诱导剂组；A549细胞和A549/Gb分为A549组、A549/Gb组、A549/Gb+槲皮素组、A549/Gb+EMT诱导 剂组、miR-101-inhibitor组、槲皮素+miR-101-inhibitor组、miR-NC组；实时荧光定量PCR（qPCR）检测各组miR-101 表达水平；Western blot法检测EZH2、转化生长因子（TGF）-β1、果蝇母亲DPP同源物4（SMAD4）、p-SMAD4和EMT标 志蛋白E钙黏蛋白（E-cadherin）、N钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）表达；CCK-8法检测各组细胞光密度 （OD）值，并计算半数抑制浓度（IC50）及耐药指数（IR）。结果 槲皮素对A549及A549/Gb的IC50分别为64、128 μmol/L。 EMT诱导剂增强A549细胞EMT特性（N-cadherin及Vimentin表达升高，E-cadherin表达降低）后，A549细胞miR-101 表达降低，EZH2、TGF-β1、p-SMAD4/SMAD4蛋白表达水平，IC50及IR升高（P＜0.05）；槲皮素可抑制A549细胞EMT 特性、降低IC50及IR，并上调miR-101，抑制EZH2、TGF-β1、p-SMAD4/SMAD4蛋白表达（P＜0.05）；EMT诱导剂可逆 转槲皮素的上述作用（P＜0.05）。与 A549 细胞相比，A549/Gb 细胞的 EMT 特性增强，IC50、IR、EZH2、TGF-β1、pSMAD4/SMAD4表达均升高（P＜0.05）；抑制miR-101或EMT 诱导剂处理，均可进一步增强A549/Gb 细胞的EMT 特 性，升高IC50、IR，上调EZH2、TGF-β1、p-SMAD4/SMAD4蛋白表达（P＜0.05）；槲皮素可抑制A549/Gb细胞的EMT进 程、降低其IC50、IR及EZH2、TGF-β1、p-SMAD4/SMAD4蛋白表达（P＜0.05）；抑制miR-101表达可逆转槲皮素的上述 作用（P＜0.05）。结论 槲皮素可通过上调miR-101，抑制EZH2表达，进而抑制EMT进程，降低NSCLC细胞耐药性。

关键词: 槲皮素, 癌, 非小细胞肺, A549细胞, 抗药性, 肿瘤, 微RNAs, 上皮-间质转化, Zeste同源蛋白2增强子, 微小RNA-101, 吉西他滨

Abstract: Objective To investigate the effect of quercetin on epithelial-mesenchymal transdifferentiation (EMT) of non-small cell lung cancer (NSCLC) and its molecular mechanism in improving gemcitabine (Gb) resistance from the perspective of microRNA-101 (miR-101)/Zeste gene homologous protein 2 (EZH2) axis. Methods A549 and A549/Gb cells were cultured with different concentrations of quercetin, and CCK-8 method was used to screen suitable quercetin concentration for the follow-up test. A549 cells were divided into the blank group, the A549 + EMT inducer group, the A549+ quercetin group and the quercetin + EMT inducer group. A549 cells and A549/Gb were divided into the A549 group, the A549/Gb group, the A549/Gb + quercetin group, the A549/Gb + EMT inducer group, the miR-101-inhibitor group, the quercetin+miR-101-inhibitor group and the miR-NC group. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression level of miR-101 in each group. The expression of EZH2, transforming growth factor (TGF) - β1/ drosophila, mother DPP homolog 4 (SMAD4), p-SMAD4, and EMT marker proteins E-cadherin, N-cadherin and Vimentin were detected by Western blot assay. The optical density (OD) of each group was measured by CCK-8 method, and IC50 and IR were calculated. Results The IC 50 of quercetin for A549 and A549/Gb were 64 and 128 μmol/L, respectively. After EMT inducer enhanced the EMT characteristics of A549 cells (the expression levels of N-cadherin and Vimentin were increased, the expression of E-cadherin was decreased), the expression of miR-101 was decreased, EZH2, TGF-β1 and pSMAD4/SMAD4 protein expression, IC50 and IR increased (P＜0.05). Quercetin could inhibit the EMT characteristics of A549 cells, reduce IC50 and IR, up-regulate miR-101, inhibit EZH2, TGF-β1 and p-SMAD4/SMAD4 protein expression (P＜0.05). EMT inducers could reverse the above effects of quercetin (P＜0.05). Compared with A549 cells, the EMT characteristics increased in A549/Gb cells, and IC50, IR, EZH2, TGF- β1 and p-SMAD4/SMAD4 expressions were all increased (P＜0.05). The inhibition of miR-101 or treatment with EMT inducer could further increase the EMT characteristics, IC50, IR, EZH2, TGF-β1 and p-SMAD4/SMAD4 protein expression of A549/Gb cells (P＜0.05). Quercetin could inhibit the EMT process of A549/Gb cells and reduce its IC50, IR and EZH2, TGF-β1 and p-SMAD4/SMAD4 protein expression (P＜0.05). The inhibition of miR-101 expression could reverse the above effects of quercetin (P＜0.05). Conclusion Quercetin can up-regulate miR-101 and inhibit EZH2 expression, thereby inhibiting EMT process and reducing NSCLC cell drug resistance.

Key words: quercetin, carcinoma, non-small-cell lung, A549 cells, drug resistance, neoplasm, microRNAs, epithelialmesenchymal transition, enhancer of Zeste homolog 2 protein, microRNA-101, gemcitabine