乐伐替尼与瑞格菲尼对肝癌细胞PD-L1的作用及机制  

Effects and Mechanism of Lenvatinib and Regorafenib on PD-L1 in Liver Cancer Cells

作　　者：许静 喻超 杨盛力 孙诚谊 XU Jing;YU Chao;YANG Shengli;SUN Chengyi(Department of Hepatobiliary Surgery,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Key Laboratory of Liver,Gallbladder,Pancreas and Spleen of Guizhou Medical University,Guiyang 550004,Guizhou,China;Guizhou Institute of Hepatobiliary,Pancreatic and Spleen Diseases,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学附属医院肝胆外科,贵州贵阳550004 [2]贵州医科大学肝胆胰脾重点实验室,贵州贵阳550004 [3]贵州省肝胆胰脾疾病研究所,贵州贵阳550004

出　　处：《贵州医科大学学报》2020年第11期1275-1282,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81860505,81860506)。

摘　　要：目的:探究乐伐替尼与瑞格菲尼在肝癌细胞中对程序性细胞死亡配体1(PD-L1)作用的分子机制。方法:取人肝癌Hep3B、SMMC-7721细胞,分别转染转化生长因子-β1(TGF-β1)空载体、干扰质粒后的肝癌细胞培养,采用实时荧光定量PCR(qRT-PCR)检测细胞中缺氧诱导因子-1α(HIF 1α)、髓细胞增生原癌基因(MYC)、缺氧诱导因子-2α(HIF 2α)、信号传导与转录激活因子3(STAT 3)、黏蛋白1-C(MUC 1-C)、细胞周期素依赖蛋白激酶5(CDK 5)、丝裂原活化蛋白激酶(MAPK 14)、表皮生长因子受体(EGFR)、间变性淋巴瘤激酶(ALK)及TGF-β1的mRNA表达,采用Western blot实验检测各组肝癌细胞中PD-L1和TGF-β1的蛋白表达量;取4周龄雌性裸鼠(BALB/c)小鼠30只,人肝癌Hep3B细胞皮下接种于小鼠右侧腹肋下,1周后将皮下成瘤的小鼠随机均分为对照组(DMSO)、乐伐替尼组(注射乐伐替尼)及瑞格菲尼组(注射瑞格菲尼),于末次给药后24 h处死小鼠,采用Western blot实验检测各组小鼠皮下瘤组织PD-L1和TGF-β1的蛋白表达量。结果:与对照组比较,乐伐替尼组和瑞格菲尼组小鼠皮下肝癌移植瘤组织中HIF 1α、MYC、HIF 2α及MUC 1-C mRNA表达均上调,STAT 3、CDK 5、MAPK14、EGFR及ALK mRNA表达均下调,但乐伐替尼组TGF-β1 mRNA表达增加,瑞格菲尼组TGF-β1 mRNA表达降低(P<0.05);与对照组比较,乐伐替尼组肝癌细胞Hep3B和SMMC-7721的TGF-β1 mRNA、PD-L1和TGF-β1蛋白表达均上升,瑞格菲尼组的TGF-β1 mRNA、PD-L1及TGF-β1蛋白表达均下降(P<0.05);肝癌细胞Hep3B和SMMC-7721干扰组TGF-β1 mRNA、PD-L 1 mRNA、TGF-β1及PD-L1蛋白表达分别较对照组降低(P<0.05)。结论:乐伐替尼使肝癌细胞PD-L 1表达上调,瑞格菲尼使PD-L 1表达下调,其机制可能是通过影响TGF-β1的表达。Objective:To investigate the molecular mechanism of lenvatinib and regorafenib on programmed cell death ligand 1 in liver cancer cells.Methods:Human liver cancer cells Hep3B and SMMC-7721 were cultured and transfected with TGF-β1 empty vector and interfering plasmid respectively;the expression of HIF1α,MYC,HIF2α,STAT3,MUC1-C,CDK5,MAPK14,EGFR,ALK,and TGF-β1 were detected by qRT-PCR;the protein expression of PD-L1 and TGF-β1 were detected by Western blot;304-week-old female mice(BALB/c)were inoculated with human liver cancer Hep3B cells under the right ventral rib,and the subcutaneous tumor-forming mice were randomly divided into control groups(DMSO),lenvatinib group(injecting with lenvatinib)and regorafenib group(injecting with regorafenib)one week later.Mice were sacrificed 24 hours after the last dose.Western blot was used to detect the protein expression of PD-L1 and TGF-β1 in the subcutaneous tumor tissues of mice in each group.Results:Compared with the control group,the mRNA expressions of HIF1α,MYC,HIF2α,and MUC1-C were up-regulated in the subcutaneous liver cancer xenograft tissues of mice in experiment groups;the mRNA expressions of STAT3,CDK5,MAPK14,EGFR,and ALK were all down-regulated,but the mRNA expression of TGF-β1 increased in the lenvatinib group,and the mRNA expression of TGF-β1 decreased in the regorafenib group(P<0.05);compared with the control group,the mRNA expressions of TGF-β1,protein expression of TGF-β1,PD-L1 were all increased in the liver cancer cells Hep3B and SMMC-7721 in the lenvatinib group,and the expression of TGF-β1 mRNA,PD-L1 and TGF-β1 protein expression of the regorafenib group all decreased(P<0.05);expressions of TGF-β1 mRNA,PD-L1 mRNA,TGF-β1 and PD-L1 protein in liver cancer cells Hep3B and SMMC-7721 interference group were lower than the control group(P<0.05).Conclusions:In liver cancer cells,lenvatinib up-regulated the expression of PD-L1,while regorafenib down-regulated the expression of PD-L1.The mechanism may be caused by affecting the expression of TGF-β

关 键 词：癌 肝细胞 乐伐替尼 瑞格菲尼 细胞程式死亡-配体1 转化生长因子-Β1 耐药 

分 类 号：R735.7[医药卫生—肿瘤]