外泌体靶向运载整合素αv/β3的小干扰RNA对未分化甲状腺癌细胞增殖、迁移和侵袭的影响  

Effects of exosomes targeted delivery of integrin αv/β3 small interfering RNA on proliferation, migration, and invasion of anaplastic thyroid carcinoma cells

作　　者：张文新[1] 李宁[1] 王深[1] 常鹏鹏 李伟健 王慧[1] 贾强[1] 谭建[1] 张瑞国[1] Zhang Wenxin;Li Ning;Wang Shen;Chang Pengpeng;Li Weijian;Wang Hui;Jia Qiang;Tan Jian;Zhang Ruiguo(Department of Nuclear Medicine,Tianjin Medical University General Hospital,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院核医学科,天津300052

出　　处：《中华实验外科杂志》2024年第1期44-48,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81801732)。

摘　　要：目的探讨通过外泌体靶向运载整合素αv/β3小干扰(si)RNA(si-αv/β3)对未分化甲状腺癌(ATC)细胞的增殖、迁移和侵袭能力的影响。方法采用蛋白质印迹法(Western blot)检测整合素αv/β3在ATC细胞系(8505C、Hth7)及人正常甲状腺细胞(上海生命科学院细胞所)中的表达;将含内化精氨酸-甘氨酸-天冬氨酸多肽(iRGD)的质粒转染人源胚胎肾细胞HEK-293T后采用超高速离心法得到靶向外泌体,采用PKH26染色验证其靶向性;进一步通过转染获得靶向运载si-αv/β3的外泌体iRGD-exos-si-av/β3(载siRNA组),将其同8505C细胞共孵育后,采用荧光定量聚合酶链反应(qPCR)和Western blot检测载siRNA组与空载外泌体组8505C细胞内整合素αv/β3的mRNA和蛋白表达水平。采用细胞计数试剂盒(CCK-8)、划痕实验和Transwell检测8505C细胞的增殖、迁移及侵袭能力。两样本间比较采用t检验,多组间比较采用单因素方差分析。结果整合素αv/β3在8505C及Hth7细胞中的表达量显著高于人正常甲状腺细胞(αv:1.04±0.12、0.90±0.11比0.31±0.01,F=50.11,P<0.01;β3:0.93±0.23、0.89±0.16比0.33±0.27,F=6.81,P<0.05)。iRGD靶向外泌体在细胞中的荧光信号明显高于非靶向外泌体组(47.18±10.39比5.64±1.43,t=6.86,P<0.05)。转染si-αv/β3至8505C细胞后,mRNA水平表达显著低于对照组(αv:0.20±0.01比1.21±0.14,t=12.49,P<0.05;β3:0.21±0.02比1.01±0.18,t=7.90,P<0.05);蛋白表达水平亦显著低于对照组(αv:0.45±0.03比0.88±0.06,t=10.33,P<0.05;β3:0.48±0.09比1.10±0.11,t=9.02,P<0.05)。iRGD-exos-si-av/β3与空载外泌体组分别与8505C细胞共孵育后,前者mRNA水平表达明显低于空载组(αv:0.30±0.08比1.05±0.02,t=16.75,P<0.05;β3:0.27±0.05比1.29±0.20,t=8.71,P<0.05);蛋白水平表达亦低于对照组(αv:0.60±0.30比1.20±0.23,t=5.72,P<0.05;β3:0.24±0.11比1.26±0.14,t=16.23,P<0.05)。载si-αv/β3的靶向外泌体组的细胞增殖率在不同时间点均明显低于空载组[载si-αv组:12Objective To explore the effects of exosomes targeted delivery of integrinαv/β3 small interfering RNA(si-αv/β3)on the proliferation,migration,and invasion of anaplastic thyroid carcinoma(ATC)cells.Methods The expression levels of integrinαv/β3 in ATC cell lines(8505C,Hth7)and human normal thyroid cells(Shanghai Institutes of Cell Biology for Biological Sciences)were detected by Western blotting.We transfected internalized arginine-glycine-aspartic acid peptide(iRGD)into HEK-293T cells and then obtained iRGD targeted exosomes by ultracentrifugation.The targeted ability of exosomes was validated by PKH26 staining.iRGD targeted exosomes delivering si-αv/β3(denoted as iRGD-exos-si-av/β3 group)were obtained by transfection of si-αv/β3 into iRGD-293T cells.The mRNA and protein expression levels of integrinαv/β3 in 8505C cells were detected by quantitative polymerase chain reaction(qPCR)and Western blotting.The proliferative,migratory,and invasive abilities were analyzed by cell counting kit-8(CCK-8)assay,scratch test,and Transwell assay in 8505C cells.The measurement data between groups were compared using a t-test and one-way analysis of variance was used for comparison between multiple groups.Results The expression level of integrinαv/β3 in 8505C and Hth7 cells was significantly higher than that in human normal thyroid cells(αv:1.04±0.12,0.90±0.11 vs.0.31±0.01,F=50.11,P<0.01;β3:0.93±0.23,0.89±0.16 vs.0.33±0.27,F=6.81,P<0.05).The fluorescence signal in the 8505C cells of iRGD-targeted exosomes group was significantly stronger than that of non-targeted exosomes(47.18±10.39 vs.5.64±1.43,t=6.86,P<0.05).After transfection with si-αv/β3 into 8505C cells,the mRNA expression level was significantly lower than that of the control group(αv:0.20±0.01 vs.1.21±0.14,t=12.49,P<0.05;β3:0.21±0.02 vs.1.01±0.18,t=7.90,P<0.05),and the protein expression was also significantly lower than that of the control group(αv:0.45±0.03 vs.0.88±0.06,t=10.33,P<0.05;β3:0.48±0.09 vs.1.10±0.11,t=9.02,P<0.05).The m

关 键 词：外泌体 甲状腺癌 整合素αv/β3 小干扰RNA 

分 类 号：R736.1[医药卫生—肿瘤]