阿伐麦布通过SOAT1重塑胆固醇代谢抑制肾透明细胞癌增殖的研究  

Inhibition of renal clear cell carcinoma proliferation by avasimibe through SOAT1-mediated cholesterol metabolism remodeling

作　　者：胡毅锋 王雁良 徐勤 焦晓静 刘子洋 王宁 王莎莎 阎磊 邵凤民 滕柳 HU Yifeng;WANG Yanliang;XU Qin;JIAO Xiaojing;LIU Ziyang;WANG Ning;WANG Shasha;YAN Lei;SHAO Fengmin;TENG Liu(Department of Nephrology,Peoples Hospital of Zhengzhou University,Henan Provincial People's Hospital,Henan Provincial Key Laboratory of Kidney Disease and Immunology,Henan Provincial Clinical Research Center for Kidney Disease,Zhengzhou,Henan 450003,China;Graduate School of Xinriang Medical University,Xinriang,Henan 453000,China)

机构地区：[1]郑州大学人民医院,河南省人民医院肾内科,河南省肾脏病免疫重点实验室,河南省肾病临床医学研究中心,河南郑州450003 [2]新乡医学院研究生院,河南新乡453000

出　　处：《中华肿瘤防治杂志》2024年第17期1051-1058,1066,共9页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(82303099);河南省科技攻关项目(222102520014,212102310201);河南省医学科技攻关计划省部共建青年项目(SBGJ202103022,SBGJ202303004)。

摘　　要：目的筛选有效抑制肾透明细胞癌(ccRCC)增殖的小分子抑制剂,探讨阿伐麦布通过其作用靶标甾醇O-酰基转移酶1(SOAT1)对ccRCC的抑制作用。方法采用细胞计数试剂盒8(CCK8)法筛选脂代谢化合物库中能够显著抑制ccRCC细胞活力的小分子抑制剂,测定对照组、敲低/过表达SOAT1组ccRCC细胞活力变化。总胆固醇和胆固醇酯荧光测定试剂盒检测对照组、阿伐麦布组及敲低SOAT1组ccRCC细胞内胆固醇酯含量;实时荧光定量聚合酶链式反应(RT-qPCR)检测SOAT1 mRNA表达量变化;蛋白质印迹法检测SOAT1表达量;平板克隆形成实验检测细胞克隆形成变化。2组比较采用独立样本t检验。结果在2轮药物筛选中,与对照组相比(1.00),阿伐麦布组769-P(第1轮:0.01±0.02,t=75.50,P<0.001;第2轮:0.02±0.00,t=348.67,P<0.001)与Caki-1(第1轮:0.53±0.01,t=62.28,P<0.001;第2轮:0.02±0.00,t=440.25,P<0.001)细胞活力均降低。阿伐麦布在ccRCC中的半数抑制浓度(IC_(50))测定结果显示,769-P(IC_(50)为43.94μmol/L)、Caki-1(IC_(50)为48.01μmol/L)较HK-2(IC_(50)为49.65μmol/L)、ACHN(IC_(50)为55.36μmol/L)、786-O(IC_(50)为56.29μmol/L)和A498(IC_(50)为61.75μmol/L)细胞对阿伐麦布更敏感,P<0.001。与对照组相比,阿伐麦布组中769-P[(855.27±17.49)μmol/L vs(798.97±15.49)μmol/L,t=4.17,P=0.014]和Caki-1[(825.77±10.10)μmol/L vs(665.88±32.40)μmol/L,t=8.16,P=0.001]细胞内胆固醇酯含量均减少。与对照组(1.00±0.05)相比,敲低SOAT1显著抑制SOAT1 mRNA在769-P(0.46±0.02)和Caki-1(0.33±0.02)细胞中的表达,差异有统计学意义,t值分别为17.15和20.39,均P<0.001;与对照组(1.00±0.00)相比,SOAT1蛋白表达量在769-P(0.38±0.14)和Caki-1(0.41±0.07)中均降低,差异有统计学意义,t值分别为7.95和15.26,P值分别为0.001和<0.001。敲低SOAT1后,与对照组(1.00)相比,769-P(0.63±0.06)和Caki-1(0.58±0.07)细胞增殖能力降低,差异有统计学意义,t值分别为10.54和10.95,均P<0.001;克隆形成能力(769-PObjective To identify small molecule inhibitors which inhibit the cell proliferation of clear cell renal cell carcinoma(ccRCC),and to clarify the role of avasimibe in inhibiting cell proliferation by targeting SOAT1 in ccRCC.Methods Cell counting kit-8(CCK8)method was used to screen small molecular inhibitors which could significantly inhibit the cell viability of ccRCC in the Lipid Metabolism Compound Library,and the changes of the cell viability of ccRCC in the control group and the SOAT1 knock-down/over-expression group were determined.The total cholesterol and cholesterol ester fluorescence determination kit was used to detect the content of cholesterol ester in ccRCC cells of control group,avasimibe group and SOAT1 knock-down group;quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect the changes of SOAT1 mRNA expression;western blot was used to detect the expression of SOATl;plate clone formation experiment was used to detect the changes of cell clone formation.The independent sample t test was used for comparison between the two groups.Results In the two rounds of drug screening,compared with the control group(1.00),the viability of 769-P(round 1:0.01±0.02,t=75.50,P<0.001;round 2:0.02±0.00,t=348.67,P<0.001)and Caki-1(round 1:0.53±0.01,t=62.28,P<0.001;round 2:0.02±0.00,t=440.25,P<0.001)cells in avasimibe group decreased.The results of 50%inhibition concentration determination of avasimibe in ccRCC showed that 769-P(IC_(50):43.94μmol/L)and Caki-1(IC_(50):48.01μmol/L)were more sensitive to avasimibe than HK-2(IC_(50):49.65μmol/L),ACHN(IC_(50):55.36μmol/L),786-O(IC_(50):56.29μmol/L)and A498(IC_(50):61.75μmol/L),P<0.001.Compared with the control group,the content of cholesterol ester in 769-P[(855.27±17.49)μmol/L vs(798.97±15.49)μmol/L,t=4.17,P=0.014]and Caki-1[(825.77±10.10)μmol/L vs(665.88±32.40)μmol/L,t=8.16,P=0.001]cells in avasimibe group decreased.Compared with the control group(1.00±0.05),knocking down SOAT1 significantly inhibited the expression of SOAT1 mRNA in

关 键 词：肾透明细胞癌 阿伐麦布 甾醇O-酰基转移酶1 胆固醇代谢 

分 类 号：R737.11[医药卫生—肿瘤]