Leptin基因沉默对胆囊癌GBC-SD和OCUG细胞系侵袭和迁移能力的影响  

The influence of silencing leptin gene on the invasion and migration abilities of gallbladder cancer GBC-SD and OCUG cells

作　　者：石万红 邹雷[1] 康强[2] 王峻峰[3] 白建华[1] 晋云 张杰[1] 张小文[2] SHI Wanhong;ZOU Lei;KANG Qiang;WANG Junfeng;BAI Jianhua;JIN Yun;ZHANG Jie;ZHANG Xiaowen(Department of Organ Transplantation,First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan Province,China;Department of Hepatobiliary Surgery,Second Affiliated Hospital of Kunming Medical University,Kunming 650101,Yunnan Province,China;Department of Hepatobiliary Surgery,First People’s Hospital of Yunnan Province,Kunming 650021,Yunnan Province,China)

机构地区：[1]昆明医科大学第一附属医院器官移植科,云南昆明650032 [2]昆明医科大学第二附属医院肝胆外科,云南昆明650101 [3]云南省第一人民医院肝胆外科,云南昆明650021

出　　处：《中国癌症杂志》2021年第11期1050-1057,共8页China Oncology

基　　金：云南省科技厅基础研究面上项目(202001AT070018);国家自然科学基金(81760430);云南省医学学科后备人才项目(H-2017037);云南省卫生和计划生育委员会医学学科带头人培养计划(D-201658);云南省科技惠民专项(2016RA011);云南省科技计划项目-科技入滇专项(2018IB007)。

摘　　要：背景与目的:研究表明,与肥胖症密切相关的源自脂肪细胞的细胞因子瘦素(leptin)在癌变和肿瘤发生中起着重要作用。通过体外实验观察leptin对人胆囊癌细胞系GBC-SD和OCUG侵袭和迁移能力的影响,探讨其可能的作用机制。方法:将胆囊癌GBC-SD和OCUG细胞分为对照组和实验组。实验组利用小干扰RNA(small interfering RNA,siRNA)技术靶向沉默胆囊癌GBC-SD和OCUG细胞的leptin表达,应用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测转染效率,通过细胞划痕实验和transwell小室法检测胆囊癌GBC-SD和OCUG细胞的迁移和侵袭能力,采用免疫荧光和免疫细胞化学实验检测siRNA干扰后胆囊癌GBC-SD和OCUG细胞的leptin蛋白水平,利用蛋白质印迹法(Western blot)检测干扰后GBC-SD和OCUG细胞的leptin和p-AKT蛋白表达。结果:细胞划痕实验结果显示,利用siRNA靶向沉默leptin后GBC-SD和OCUG细胞的迁移能力下降(t=26.614,P<0.01;t=19.338,P<0.01)。Transwell迁移实验结果显示,利用siRNA靶向沉默leptin后GBC-SD和OCUG细胞的迁移能力也下降(t=7.185,P=0.002;t=8.889,P=0.003)。Transwell侵袭实验结果显示,干扰后GBC-SD和OCUG细胞的侵袭能力下降(t=10.183,P=0.001;t=9.697,P=0.001)。免疫荧光和免疫细胞化学实验结果显示,干扰后GBC-SD和OCUG细胞的leptin蛋白表达下降,Western blot检测结果表明,靶向沉默GBC-SD细胞后leptin和p-AKT蛋白表达下调(t=26.463,P<0.01;t=13.904,P<0.01),靶向沉默OCUG细胞后leptin和p-AKT蛋白表达也下调(t=21.335,P<0.01;t=17.914,P<0.01)。结论:下调GBC-SD和OCUG细胞的leptin表达可抑制细胞侵袭和转移能力,并可能通过AKT信号通路调控胆囊癌细胞的侵袭和迁移。Leptin有望成为胆囊癌治疗的一个重要靶点。Background and purpose:Emerging evidence suggests that fat cell-derived cytokine leptin,which is closely related to obesity,plays an important role in carcinogenesis and tumorigenesis.In this study,the effect of leptin on the invasion and migration abilities of GBC-SD and OCUG cells were observed in vitro,and its possible related mechanisms were explored.Methods:GBC-SD and OCUG cells were divided into control group and experimental group.In the experimental group,we used small interfering RNA(siRNA)to target and silence the expression of leptin in GBC-SD and OCUG cells.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to detect the transfection efficiency.Wound healing assay and transwell assay were used to detect the migration and invasion abilities of GBC-SD and OCUG cells.The immunofluorescence and immunocytochemistry experiments were used to detect the expression of leptin protein in GBC-SD and OCUG cells after siRNA interference.The protein expressions of leptin and p-AKT in GBC-SD and OCUG cells after interference were detected by Western blot.Results:The results of wound healing assay showed that the migration abilities of GBC-SD and OCUG cells decreased after silencing leptin with siRNA interference(t=26.614,P<0.01;t=19.338,P<0.01).The results of transwell migration assay also showed that the migration abilities of GBC-SD and OCUG cells decreased after silencing leptin with siRNA interference(t=7.185,P=0.002;t=8.889,P=0.003).Transwell invasion assay results showed that the invasion abilities of GBC-SD and OCUG cells decreased after interference(t=10.183,P=0.001;t=9.697,P=0.001).Immunofluorescence and immunocytochemistry experiments showed decreased leptin protein expression in GBC-SD and OCUG cells after interference.Western blot showed that leptin and p-AKT protein expressions were down-regulated after targeted silencing in GBC-SD cells(t=26.463,P<0.01;t=13.904,P<0.01).Western blot showed that leptin and p-AKT protein expressions were also down-regulated after targeted silen

关 键 词：胆囊癌 瘦素 侵袭 迁移 

分 类 号：R735.8[医药卫生—肿瘤]