L型氨基酸转运载体1表达下调抑制结直肠癌细胞HT29的增殖和迁移能力  

Knockdown of L-type amino acid transporter 1 inhibits the proliferation and migration of colorectal cancer cell line HT29

作　　者：杨张朔 王昕[2] 杨铁成 黄牛[2] 孔秀英[2] 黄智民[2] 卢作鹏[2] 习一清 冯茂辉[1] Yang Zhangshuo;Wang Xin;Yang Tiecheng;Huang Niu;Kong Xiuying;Huang Zhiming;Lu Zuopeng;Xi Yiqing;Feng Maohui(Departmentof Gastrointestinal Surgery,Zhongnan Hospital,Clinical Cancer Study Center of Hubei Provence,Key Laboratory of Tumor Biological Behavior of Hubei Provence,Clinical Medical Research Center of Peritoneal Cancer of Wuhan,Wuhan University,Wuhan 430071,China;Departmentof Gastrointestinal Surgery,Tongcheng People’s Hosptial,Tongcheng 437400,China)

机构地区：[1]武汉大学中南医院胃肠外科武汉市腹膜癌临床医学研究中心湖北省肿瘤医学临床研究中心肿瘤生物学行为湖北省重点实验室,430071 [2]通城县人民医院胃肠外科,湖北通城437400

出　　处：《中华实验外科杂志》2019年第10期1795-1797,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81770283);湖北省卫生与计划生育委员会科研基金(WJ2017M249);武汉市腹膜癌临床医学研究中心资助项目(2015060911020462).

摘　　要：目的观察L型氨基酸转运载体1(LAT1)对结直肠癌细胞增殖和迁移能力的影响.方法设计并合成靶向LAT1的小干扰RNA(siLAT1)和阴性对照小干扰RNA(siCON),并在结直肠癌细胞株HT29中进行转染,48 h后利用实时定量反转录聚合酶链反应(RT-qPCR)、蛋白质印迹法(Western blot)检测目的基因mRNA和蛋白水平的表达,进行敲降效率的验证.利用细胞计数试剂盒(CCK-8)实验检测干扰组(siLAT1组)和阴性对照组(siCON组)细胞增殖,利用Transwell实验检测siLAT1组和siCON组细胞的迁移能力.结果 siLAT1和siCON瞬时转染HT29细胞48 h后,siLAT1对HT29细胞LAT1 mRNA表达的抑制率为(73.2±5.2)%,差异有统计学意义(t=11.410,P<0.01).Western blot结果进一步证实靶向LAT1的小干扰RNA(siRNA)的敲减效率(0.68±0.06比0.21±0.04,t=4.492,P<0.05).CCK-8实验表明LAT1基因敲降后的细胞株HT29增殖能力弱于阴性对照组(P<0.01).Transwell迁移实验表明在转染后siLAT1组迁移细胞数明显小于阴性对照组(66 ±11比156±29,t=5.083,P<0.05).结论 LAT1表达下调抑制了结直肠癌肿瘤细胞的增殖和迁移过程.Objective To investigate the effect of L-type amino acid transporter 1 (LAT1) on colorectal cancer cell proliferation and migration. Methods LAT1-specific small interfering RNA (siLAT1) and control small interfering RNA (siCON) were synthesized. Then, HT29 cell line was selected to generate the colorectal cancer cell model with LAT1 deficiency using small interfering RNA (siRNA) transfection. real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to confirm the efficiency of the transfection after treatment with siRNA for 48 h. After that, cell counting kit-8 (CCK-8) assay and Transwell migration assay were used to examine whether silencing LAT1 with siRNA could affect cell proliferation and migration in HT29 cells, respectively. Results Silencing LAT1 with siRNA could drastically reduce the expression of LAT1 mRNA and the Knockdown efficiency was (73.2±5.2)%(t=11.410, P<0.01). Meanwhile, Western blotting also comfirmed that siLAT1 could reduce protein expression of LAT1 (0.68±0.06 versus 0.21±0.04, t=4.492, P<0.05). Furthermore, CCK-8 assay suggested that knockdown of LAT1 significantly inhibited the viability of HT29 cells treated with siLAT1 versus those treated with siCON (P<0.05). The results of the Transwell migration assay indicated that migration of HT29 cells treated with siLAT1 was drastically reduced compared with those treated with siCON (66±11 versus 156±29, t=5.083, P<0.05). Conclusion Downregulated LAT1 suppressed the ability of colorectal cell proliferation and migration in vitro.

关 键 词：L型氨基酸转运载体1 结直肠癌 小干扰RNA 增殖 迁移 

分 类 号：R735.3[医药卫生—肿瘤]