恩格列净通过雷帕霉素靶点蛋白抑制胃癌的分子机制初探  

Preliminary Investigation of the Molecular Mechanism of Empagliflozin Suppressing Gastric Cancer Through Mammalian Target of Rapamycin

作　　者：饶慧玲 程旺 于娟 安晓彤 邓浩君 张昭阳 武福云[1] 戢福云 李珊[1] RAO Huiling;CHENG Wang;YU Juan;AN Xiaotong;DENG Haojun;ZHANG Zhaoyang;WU Fuyun;JI Fuyun;LI Shan(Institute of Basic Medical Sciences,Hubei University of Medicine,Shiyan 442000,China;Department of Medical Engineering,The First Affiliated Hospital of Army Medical University,Chongqing 400038,China)

机构地区：[1]湖北医药学院基础医学研究所,十堰442000 [2]陆军军医大学第一附属医院医学工程科,重庆400038

出　　处：《四川大学学报（医学版）》2023年第6期1146-1153,共8页Journal of Sichuan University(Medical Sciences)

基　　金：湖北医药学院自由探索项目(No.FDFR201901);湖北省教育厅科技项目(No.Q20212103);基础医学院研究生科技创新项目(No.JC202109);大学生创新创业训练项目(No.S202110929011)资助。

摘　　要：目的 恩格列净(empagliflozin, EMPA)是钠-葡萄糖转运蛋白2(sodium-glucose cotransporter 2, SGLT2)特异性抑制剂。通过综合网络药理学预测EMPA对胃腺癌的干预靶点,并利用细胞生物学和分子生物学实验对其作用及分子机制进行验证。方法 使用生物信息学分析胃腺癌预后与SGLT2表达情况的相关性,网络药理学分析EMPA和胃腺癌的共同靶点。用不同浓度的EMPA孵育人胃腺癌细胞AGS 24 h后,CCK8法检测细胞增殖率;选择0、3、6 mmol/L EMPA孵育AGS细胞,实时细胞分析(real-time cell analysis, RTCA)和EdU(5-ethynyl-2-deoxyuridine)检测EMPA对胃腺癌细胞增殖的抑制能力,伤口愈合实验和Transwell实验检测EMPA对胃腺癌细胞迁移和侵袭的抑制能力,Western blot检测雷帕霉素(mammalian target of rapamycin, mTOR)和磷酸化mTOR(phosphorylated mTOR, p-mTOR)表达。BALB/c(nu/nu)裸鼠均于腋下种植5×10~6 AGS细胞,分为对照组、低剂量组和高剂量组,每组7只,1周后,对照组每日腹腔注射生理盐水,低剂量组和高剂量组每日腹腔注射EMPA 3 mg/kg和5 mg/kg,给药1周后检测肿瘤体积。结果 低表达SGLT2的胃腺癌患者生存期和存活率均高于高表达SGLT2的胃腺癌患者。收集了104个EMPA相关潜在靶点和2 028个胃腺癌相关靶点,胃腺癌相关的45个靶点和EMPA潜在靶点相重合,从中鉴定出10条相关信号通路和4个核心基因。这4个关键基因分别是细胞周期依赖激酶4基因(cyclin-dependent kinase-4, CDK4)、3-磷酸甘油醛脱氢酶基因(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、雷帕霉素靶点蛋白基因(mammalian target of rapamycin, mTOR)和周期蛋白E1基因(cyclin E1, CCNE1)。CCK-8检测结果示0.39~50 mmol/L EMPA能抑制AGS细胞增殖;与对照组相比,RTCA结果表明3 mmol/L、6 mmol/L EMPA组细胞生长曲线下移。与对照组相比,EdU检测发现3 mmol/L、6 mmol/L EMPA能够抑制AGS细胞的增殖(P<0.05),伤口愈合实验和Transwell实验结果表明,3 mmol/LObjective To predict the intervention targets of empagliflozin(EMPA),a specific inhibitor of sodium-glucose cotransporter 2(SGLT2),in gastric adenocarcinoma through comprehensive network pharmacology,and to validate the effects and the molecular mechanisms of EMPA through cellular and molecular biology experiments.Methods Bioinformatics analysis of gastric adenocarcinoma was conducted to assess the correlation between gastric adenocarcinoma prognosis and SGLT2 expression.Network pharmacology was utilized to identify shared targets of EMPA and gastric adenocarcinoma.AGS cells,a human gastric adenocarcinoma cells line,were incubated with EMPA at different concentrations for 24 h and,then,cell proliferation was assessed using the CCK8 assay.After AGS cells were incubated with EMPA at the doses of 0,3,and 6 mmol/L,real-time cell analysis(RTCA)and 5-ethynyl-2-deoxyuridine(EdU)incorporation were used to evaluate EMPA's inhibitory effects on the proliferation of the AGS cells.In addition,wound healing and Transwell assays were performed to assess the inhibitory effect of EMPA on the migration and invasion of the APC cells and Western blot analysis was conducted to examine the expression of mammalian target of rapamycin(mTOR)and phosphorylated mTOR(p-mTOR).BALB/c(nu/nu)nude mice were implanted with 5×106 AGS cells in the axilla.The mice were divided into three groups,a control group,a low-dose group,and a high-dose group,each consisting of 7 mice.After one week,the control group received daily intraperitoneal injections of normal saline,while the low-dose group and high-dose group received daily intraperitoneal injections of EMPA at the doses of 3 mg/kg and 5 mg/kg,respectively.The tumor volume was measured one week after the drug intervention started.Results Gastric adenocarcinoma patients with low expression of SGLT2 exhibited longer survival time and higher survival rate than those with high expression of SGLT2 did.A total of 104 EMPA-related potential targets and 2028 targets associated with gastric adenocarcinoma w

关 键 词：钠-葡萄糖共转运蛋白2 恩格列净 胃腺癌 整合网络药理学 MTOR 

分 类 号：R735.2[医药卫生—肿瘤]