摘要
目的:从表观遗传学角度探讨索拉非尼(sorafenib,Sora)治疗肝细胞癌发生耐药的机制,考察表观遗传药物地西他滨(decitabine,DAC)对索拉非尼肝细胞癌敏感性的影响,为肝癌的临床治疗提供新的思路与方法。方法:利用GEPIA 2数据库检索508例原发性肝细胞肝癌(hepatocellular carcinoma,HCC)患者信息,通过Kaplan-Meier法分析有机阴离子转运多肽1B3(organic anion transporting polypeptide 1B3,OATP1B3)表达与患者生存时间的相关性;采用亚硫酸氢盐甲基化法检测基因溶质载体有机阴离子转运体1B3(solute carrier organic anion transporter 1B3,SLCO1B3)启动子的甲基化率;采用RT-qPCR,Western Blot检测DAC作用前后肝癌细胞株OATP1B3的表达变化;采用RTCA-eSight检测索拉非尼与DAC联用对肝癌细胞增殖的影响;采用LC-MS/MS检测索拉非尼与DAC联用后肝癌细胞对索拉非尼摄入量的变化。结果:GEPIA 2数据库分析结果显示,OATP1B3高表达的HCC患者总体生存率均显著高于低表达者;亚硫酸氢盐甲基化测序结果显示Hep3B,HepG2中SLCO1B3的启动子甲基化率较高;RT-qPCR,Western Blot结果表明,肝癌细胞株Hep3B,HepG2中OATP1B3的mRNA及蛋白表达相对较低,且DAC孵育后OATP1B3的表达均上调;RTCA-eSight实验结果显示,联用DAC后Hep3B,HepG2的增殖率显著低于索拉非尼组;LC-MS/MS结果显示,HEK293-OATP1B3对索拉非尼的摄入量是HEK293-Wild的2.10倍。联用DAC后,Hep3B,HepG2对索拉非尼的摄入量提高1.87和2.47倍。结论:DAC通过抑制SLCO1B3 DNA甲基化,上调OATP1B3的表达,提高转运索拉非尼的能力,增加索拉非尼在肝癌细胞中的蓄积,增强对肝癌细胞的敏感性,从而逆转索拉非尼的耐药。
Objective:To investigate the mechanism of drug resistance occurring in hepatocellular carcinoma treated with sorafeinib from epigenetic perspective,and examine the effect of sensitivity of sorafeinib on hepatocellular carcinoma after in vita combination of epigenetic drug decitabine(DAC),so as to provide new ideas and methods for clinical treatment of hepatocellular carcinoma.Methods:The GEPIA 2 database was used to retrieve information of 508 primary hepatocellular liver cancer patients,and the correlation between the expression of OATP1B3 and survival time was analyzed by Kaplan-Meier method.The methylation rate of SLCO1B3 promoter was detected by bisulfite methylation method.RT-qPCR and Western blot were used to detect the expression changes of cancer cell lines OATP1B3 before and after liver DAC treatment.RTCA-eSight experiment was performed to monitor the effect of sorafeinib in combination with DAC on the proliferation of hepatocellular carcinoma cells.Changes in the uptake of sorafeinib by hepatocellular carcinoma cells after the combination of the two drugs were detected by experimental LC-MS/MS.Results:The results of GEPIA2 database analysis showed that the overall survival rate of patients with hepatocellular carcinoma with high OATP1B3 expression was significantly higher than those with low expression.Bisulfite methylation sequencing showed that the promoter methylation rate of SLCO1B3 was higher in Hep3B and HepG2.RT-qPCR and Western blot showed that the mRNA and protein expressions of OATP1B3 in hepatocellular carcinoma cell lines Hep3B and HepG2 were relatively low,and the expression of OATP1B3 was upregulated after incubation with DAC.RTCA-eSight experiment showed that DAC combination treatment significantly enhanced the effect of sorafeinib on Hep3B and HepG2 inhibition.LC-MS/MS determination showed that the uptake of HEK293-OATP1B3 on sorafeinib was 2.10 times higher than that of HEK293-Wild.The uptake of sorafeinib by Hep3B and HepG2 was increased by 1.87-fold and 2.47-fold after being combined
作者
张苗
王泽
傅恒涛
文蕊鑫
韩巧巧
崔涛
伊秀林
闫凤英
刘昌孝
ZHANG Miao;WANG Ze;FU Heng-tao;WEN Rui-xin;HAN Qiao-qiao;CUI Tao;YI Xiu-lin;YAN Feng-ying;LIU Chang-xiao(School of Pharmacy,Anhui Medical University,Hefei 230032,China;State Key Laboratory of Drug Delivery Technology and Pharmacokinetics,Tianjin Institute of Pharmaceutical Research,Tianjin 300301,China;Tianjin Hechuang Biotechnology Co.,Ltd.,Tianjin 300301,China;Research Unit for Drug Metabolism,Chinese Academy of Medical Sciences,Beijing 100730,China;School of Pharmacy,North China University of Science and Technology,Tangshan 063210,China)
出处
《中国新药杂志》
CAS
CSCD
北大核心
2023年第16期1660-1667,共8页
Chinese Journal of New Drugs
基金
中国医学科学院医学与健康科技创新工程资助项目(2019-I2M-5-020)
天津市“项目+团队”重点培养专项资助项目(创新类)(XC202030)。
