巴马汀结合MYC G-四链体干预结肠癌细胞增殖和凋亡的作用及分子机制  

Effect of Palmatine Binding to MYC G-quadruplex on Proliferation and Apoptosis of Colon Cancer Cells and Molecular Mechanism

作　　者：温丽娜 李建辉[2] 石汉平 WEN Li-na;LI Jian-hui;SHI Han-ping(Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China;Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China)

机构地区：[1]首都医科大学附属北京世纪坛医院,北京100038 [2]中国科学院生物物理研究所,北京100101

出　　处：《中国实验方剂学杂志》2021年第21期98-104,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81803773);国家重点研发计划项目(2017YFC1309200)。

摘　　要：目的:探究巴马汀是否通过结合MYC原癌基因启动子区G-四链体干预结肠癌HCT116细胞的增殖及凋亡及其可能的分子机制。方法:采用荧光光谱分析巴马汀与MYC基因G-四链体的结合能力,采用圆二色谱分析巴马汀对MYC基因G-四链体构型的影响,采用分子对接预测二者的结合模式,采用荧光显微镜分析巴马汀分子在HCT116细胞中的定位,采用实时荧光定量聚合酶链式反应分析巴马汀对MYC基因转录的影响,采用蛋白免疫印迹法检测巴马汀对MYC蛋白表达的影响,进一步采用噻唑蓝比色法分析巴马汀对HCT116细胞活力的影响,采用流式细胞术分析巴马汀对HCT116细胞凋亡的影响。结果:荧光光谱及分子对接研究表明巴马汀可能以堆积方式与MYC基因G-四链体结合,圆二色谱分析表明马汀能够维持MYC基因G-四链体的平行构型,荧光成像分析表明巴马汀分布于细胞核中且能够与G-四链体结合。此外,巴马汀抑制了MYC基因转录及MYC蛋白表达,抑制了HCT116细胞的增殖并促进了细胞凋亡。结论:巴马汀能够结合MYC基因G-四链体并抑制其基因转录,进而抑制MYC蛋白表达,这可能是巴马汀抑制结肠癌HCT116细胞增殖并促进其凋亡的分子机制之一。Objective:To explore whether palmatine interferes with the proliferation and apoptosis of colon cancer HCT116 cells by binding to G-quadruplex in the promoter region of MYC proto-oncogene and its possible molecular mechanism.Method:Fluorescence spectrum was used to analyze the binding ability of palmatine to MYC G-quadruplex.Circular dichroism analysis was conducted to confirm the effect of palmatine on the configuration of MYC G-quadruplex,followed by the prediction of their binding mode based on molecular docking and the localization analysis of palmatine in HCT116 cells under a fluorescence microscope.The effects of palmatine on MYC gene transcription and MYC protein expression were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot,respectively.The effects of palmatine on the viability and apoptosis of HCT116 cells were further assayed by thiazolyl blue tetrazolium bromide(MTT)assay and flow cytometry.Result:As revealed by fluorescence spectrum and molecular docking analysis,palmatine might bind to G-quadruplex of MYC gene through stacking.Circular dichroism analysis showed that palmatine could maintain the parallel configuration of MYC G-quadruplex.It was discovered in fluorescence imaging that palmatine was distributed in the nucleus and bond to G-quadruplex of MYC gene.In addition,palmatine inhibited MYC gene transcription,MYC protein expression,as well as the viability of HCT116 cells,and promoted the apoptosis of HCT116 cells.Conclusion:Palmatine is able to bind to MYC G-quadruplex to further inhibit the expression of MYC gene and protein expression,which may be one of the molecular mechanisms of palmatine in suppressing the proliferation of colon cancer HCT116 cells and facilitating their apoptosis.

关 键 词：巴马汀 MYC基因 G-四链体 结肠癌 增殖 细胞凋亡 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学]