基于miR-1/HIF-1α轴研究六君子汤乙酸乙酯提取物调控食管癌细胞TE-1能量代谢的作用机制  

Mechanism of Ethyl Acetate Extract of Liujunzi Decoction(六君子汤)Regulating Energy Metabolism in Esophageal Cancer TE-1 Cells Based on miR-1/HIF-1αAxis

作　　者：陈星[1] 周哲旭 尚艺婉 刘洋[1] 刘娅茹 胡啸博 菅佳宁 肖小乔 陈玉龙[1] CHEN Xing;ZHOU Zhexu;SHANG Yiwan;LIU Yang;LIU Yaru;HU Xiaobo;JIAN Jianing;XIAO Xiaoqiao;CHEN Yulong(Henan University of Chinese Medicine,Henan Provincial Key Laboratory of Prescription and Syndrome Signal Transduction,Zhengzhou 450046,Henan,China)

机构地区：[1]河南中医药大学,河南省中医方证信号传导重点实验室,河南郑州450046

出　　处：《中华中医药学刊》2025年第4期133-139,I0029,共8页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82074313)。

摘　　要：目的探索六君子汤乙酸乙酯提取物(EAELD)能否通过miR-1/HIF-1α轴调控食管癌细胞TE-1的能量代谢及其分子机制。方法前期实验已建立稳定转染阻滞miR-1表达细胞株(IN)及空白对照(NC),采用MTT法得到EAELD对NC的半数抑制浓度(IC_(50))及30%抑制浓度(IC_(30)),将其作为六高、六低浓度分别作用于IN与NC进行对比。实时荧光定量PCR(RT-qPCR)法检测EAELD对于TE-1细胞miR-1、HIF-1αmRNA的影响。双荧光素酶基因报告实验验证miR-1、HIF-1α靶向关系。然后将实验分组为NC组、六低组、六高组、IN+六低组、IN+六高组,Seahorse能量代谢分析系统检测线粒体压力与糖酵解速率,蛋白免疫印迹法(WB)检测能量代谢相关分子蛋白表达水平变化。结果MTT结果显示EAELD能够显著抑制NC增殖能力,呈浓度梯度依赖,选取20μg/mL(IC_(30))、40μg/mL(IC_(50))为低、高浓度组。RT-qPCR结果显示EAELD高浓度组显著促进miR-1的基因表达(P<0.05),HIF-1α有下降趋势,差异无统计学意义。双荧光素酶基因报告实验显示与野生型(WT)+阴性对照(mimics NC)组相比,WT+mimics荧光强度明显下降(P<0.05),与突变型(MUT)+阴性对照(mimics NC)组相比,MUT+mimics荧光强度无明显差别。将高、低浓度EAELD作用于IN时,MTT结果显示与NC相比,EAELD对于IN的抑制作用显著降低(P<0.05)。Seahorse能量代谢结果显示与NC组相比,EAELD高、低浓度组均可以抑制线粒体耗氧与糖酵解的比值(P<0.01,P<0.05)、糖酵解潜能(P<0.01)、基础呼吸值、质子漏耗氧(P<0.01,P<0.05)、线粒体最大耗氧能力、备用呼吸能力(P<0.01),高浓度组可以抑制基础糖酵解、补偿糖酵解、非线粒体耗氧、ATP合成耗氧(P<0.01)。与NC+六低组相比,IN+六低组的基础糖酵解、线粒体与基础糖酵解的比值、糖酵解潜能(P<0.05)、ATP合成耗氧(P<0.01)显著升高;与NC+六高组相比,IN+六高基础糖酵解、补偿糖酵解(P<0.05)、基础呼吸能力、质子漏耗氧、�Objective To explore whether ethyl acetate extract of Liujunzi Decoction(EAELD)can regulate the energy metabolism of TE-1 of esophageal cancer cells via the microRNA-1(miR-1)/hypoxia inducible factor-1α(HIF-1α)axis.Methods Stable transfection blocking miR-1 expression cell lines(miR-1 inhibitor,IN)and blank controls(miR-1 NC,NC)have been established in the preliminary experiments.The half maximal inhibitory concentration(IC_(50))and 30%inhibitory concentration(IC_(30))of EAELD on NC were obtained using MTT assay,and were used as the Liujunzi Decoction high and low concentrations to compare their effects on IN and NC,respectively.The effect of EAELD on miR-1 and HIF-1αmRNA in TE-1 cells was measured by quantitative real-time PCR(RT-qPCR).Dual-luciferase gene reporter assay was used to validate the targeting relationship of miR-1 and HIF-1α.Then the experiments were divided into NC,Liujunzi Decoction low concentration,Liujunzi Decoction high concentration,IN+Liujunzi Decoction low concentration and IN+Liujunzi Decoction high concentration groups.Seahorse energy metabolism analysis system detected the mitochondrial pressure and glycolysis rate,and the protein expression level was detected by Western Blot.Results MTT results showed that EAELD could significantly inhibit the proliferation of NC in a concentration gradient dependent manner.The low and high concentration groups were 20μg/mL(IC_(30))and 40μg/mL(IC_(50)),respectively.The RT-qPCR results showed that the EAELD high concentration group significantly promoted the gene expression of miR-1(P<0.05)and HIF-1αshowed a downward trend,without statistically significant difference.Dual-luciferase gene reporter assay showed that the fluorescence intensity of wild type(WT)+mimics group was significantly lower than that of WT+negative control(mimics NC)group(P<0.05),and there was no significant difference in the fluorescence intensity of mutant(MUT)+mimics group compared with that of MUT+mimics NC group.When high and low concentrations of EAELD were applied to IN,

关 键 词：六君子汤乙酸乙酯提取物 食管癌 能量代谢 miR-1/HIF-1α轴 分子机制 

分 类 号：R289.5[医药卫生—方剂学]