Beclin1对肺癌细胞紫杉醇耐药的影响及其机制  

Effect of Beclin1 on paclitaxel resistance in lung cancer cells and its molecular mechanism

作　　者：申霖[1] 张秋峰[2] 乐艳青 焦鹏[2] Shen Lin;Zhang Qiufeng;Le Yanqing;Jiao Peng(Department of Emergency,Shangqiu First People’s Hospital,Shangqiu 476100,China;Department of EICU,Shangqiu First People’s Hospital,Shangqiu 476100,China;Department of PCCM,Guangdong Provincial People’s Hospital,Gangzhou 510080,China)

机构地区：[1]商丘市第一人民医院急诊科,476100 [2]商丘市第一人民医院急诊重症监护室,476100 [3]广东省人民医院呼吸与危重症医学科,广州510080

出　　处：《中华实验外科杂志》2022年第8期1457-1460,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨Beclin1对肺癌细胞紫杉醇耐药的影响及其分子机制。方法人非小细胞肺癌A549和小鼠LLC Lewis肺癌细胞采用梯度递增法建立紫杉醇耐药细胞株A549/DOX和LLC/DOX。采用蛋白质免疫印迹(Western blot)分析耐药和非耐药细胞株Beclin1蛋白表达水平。采用对照慢病毒和Beclin1慢病毒感染A549/DOX和LLC/DOX, 建立A549/DOX组、A549/DOX/Beclin1组、LLC/DOX和LLC/DOX Beclin1组, 采用细胞计数试剂盒(CCK-8)、5-乙炔基-2’脱氧尿嘧啶核苷(EdU)染色、克隆形成实验和体外移植瘤分析紫杉醇对4组细胞增殖的影响。采用流式细胞仪分析紫杉醇对4组细胞凋亡的影响;Western blot分析4组细胞凋亡和自噬相关蛋白表达水平。计量资料比较采用t检验。结果 A549/DOX组和LLC/DOX组细胞48 h吸光度(A)值(1.63±0.05、1.72±0.13)明显高于A549/DOX/Beclin1组和LLC/DOX Beclin1组(1.09±0.13、1.21±0.06), 差异有统计学意义(t=9.895、9.624, P<0.05)。EdU染色结果显示, A549/DOX组和LLC/DOX组细胞EdU染色阳性率[(93.71±2.81)%、(95.29±3.54)%]明显高于A549/DOX/Beclin1组和LLC/DOX Beclin1组[(62.14±7.52)%、(70.14±6.09)%], 差异有统计学意义(t=10.410、9.435, P<0.05)。克隆形成实验结果显示, A549/DOX组和LLC/DOX组细胞克隆形成数量[(115.43±10.75)、(142.57±23.71)个]明显高于A549/DOX/Beclin1组和LLC/DOX Beclin1组[(71.57±7.98)、(86.14±3.13)个], 差异有统计学意义(t=8.667、6.224, P<0.05)。A549/DOX组和LLC/DOX组细胞肿瘤体积[(823.71±44.89)、(1 144.86±106.45) mm^(3)]明显高于A549/DOX/Beclin1组和LLC/DOX Beclin1组[(585.57±41.80)、(774.14±48.95) mm3], 差异有统计学意义(t=8.667、6.224, P<0.05)。A549/DOX组和LLC/DOX组肿瘤重量[(2.53±0.27)、(2.87±0.41) g]明显高于A549/DOX/Beclin1组和LLC/DOX Beclin1组[(1.15±0.09) g、(1.46±0.13) g], 差异有统计学意义(t=12.660、8.730, P<0.05)。A549/DOX组和LLC/DOX组细胞凋亡水平[(33.16±3.31)%、(29.32±3.62)%]明显低于A549/DOX/Beclin1�Objective To investigate the effect of Beclin1 on Paclitaxel resistance in lung cancer cells and its molecular mechanism.Methods Paclitaxel resistant cell lines A549/DOX and LLC/DOX were established by gradient method in human non-small cell lung cancer A549 and mouse LLC Lewis lung cancer cells.The protein expression levels of Beclin1 in drug-resistant and non drug-resistant cell lines were detected by Western blotting.A549/dox and LLC/DOX were infected with control lentivirus and Beclin shRNA lentivirus.A549/DOX group,A549/DOX/Beclin1 group,LLC/DOX AND LLC/DOX Beclin1 group were established.The effects of paclitaxel on cell proliferation of the four groups were analyzed by cell counting kit-8(CCK-8),5-Ethynyl-2′-deoxyuridine(EdU)staining,clonogenic assay and in vitro tumor transplantation.The effect of paclitaxel on apoptosis was analyzed by flow cytometry.The expression levels of apoptosis and autophagy related proteins in the four groups were analyzed by Western blotting.The measurement data were compared by t test.Results The 48-h light absorbance(A)value in A549/DOX group(1.63±0.05)and LLC/DOX group(1.72±0.13)was significantly higher than that in A549/DOX/Beclin1 group(1.09±0.13)and LLC/DOX Beclin1 group(1.21±0.06)(t=9.895,9.624,P<0.05).The positive rate of EdU staining in A549/DOX group[(93.71±2.81)%]and LLC/DOX group[(95.29±3.54)%]was significantly higher than that in A549/DOX/Beclin1 group[(62.14±7.52)%]and LLC/DOX Beclin1 group[(70.14±6.09)%](t=10.410,9.435,P<0.05).The number of clonogenic cells in A549/DOX group(115.43±10.75)and LLC/DOX group(142.57±23.71)was significantly greater than that in A549/DOX/Beclin1 group(71.57±7.98)and LLC/DOX Beclin1 group(86.14±3.13)(t=8.667,6.224,P<0.05).The tumor volume in A549/DOX group[(823.71±44.89)mm3]and LLC/DOX group[(1144.86±106.45)mm^(3)]was significantly greater than that in A549/DOX/Beclin1 group[(585.57±41.80)mm^(3)]and LLC/DOX Beclin1 group[(774.14±48.95)mm^(3)](t=8.667,6.224,P<0.05).The tumor weight in A549/DOX group[(2.53±0.27)g]and LLC/

关 键 词：BECLIN1 肺癌 紫杉醇 耐药 凋亡 自噬 

分 类 号：R734.2[医药卫生—肿瘤]