siRNA沉默NGAL基因对结肠癌细胞行为的影响  

Effect of NGAL gene silencing by siRNA on the behavior of colon cancer cells

作　　者：王厚东 沈忠[1] 杨关根[1] 鲁振锋 陈金明 刘道成 李良飞 Wang Houdong;Shen Zhong;Yang Guangen;Lu Zhenfeng;Chen Jinming;Liu Daocheng;Li Liangfei(Department of Coloproctology, The Hangzhou No. 3 Hospital, Hangzhou Clinical School, Anhui Medical University, Hangzhou 310009, China)

机构地区：[1]安徽医科大学杭州临床学院,杭州市第三人民医院肛肠外科,浙江杭州310009

出　　处：《实用肿瘤杂志》2018年第2期122-127,共6页Journal of Practical Oncology

基　　金：浙江省科技厅公益项目(2013C33210);浙江省卫生厅骨干人才项目(2013RCB013);杭州市科技局重点专科专病项目(20140733Q20)

摘　　要：目的探讨中性粒细胞明胶酶相关载脂蛋白(neutrophils gelatinases-associated lipocalin,NGAL)的基因沉默作用对结肠癌细胞增殖、迁移和凋亡的影响。方法采用转染试剂盒ribo FECTTMCP Transfection Kit负载NGAL siRNA转染结肠癌细胞株HT-29(转染组),以未转染的HT-29细胞为对照组。实时荧光定量PCR和ELISA分别检测细胞NGAL mRNA水平和细胞培养上清液中NGAL蛋白水平,采用细胞划痕实验、Promega Cell Proliferation Assay G3580和BD C6流式细胞仪分别检测细胞迁移、增殖和凋亡情况。结果 HT-29细胞转染NGAL siRNA72 h后,转染组NGAL mRNA水平为对照组的0.123(P<0.05);转染48 h后,转染组细胞培养上清液中NGAL蛋白为(0.328±0.055)μg/L,低于对照组的(1.071±0.143)μg/L(t=8.205,P=0.001)。转染48 h后,转染组凋亡率为(18.87±1.65)%,低于对照组的(11.93±1.90)%(t=-4.752,P=0.009)。转染24 h后,转染组细胞迁移率为对照组的(23.35±6.35)%(t=11.930,P<0.01)。转染48 h后,转染组细胞增殖率为对照组的(90.00±5.30)%(t=1.983,P=0.118)。结论 NGAL siRNA可以抑制结肠癌细胞NGAL基因和蛋白的表达,抑制细胞迁移,促进细胞凋亡。Objective To explore the impact of neutrophils gelatinases-associated lipocalin（NGAL） gene silencing on the proliferation,migration and apoptosis of colon cancer cells. Methods The colon cancer cell line HT-29 was transfected with NGAL siRNA,which was encapsulated by ribo FECTTMCP Transfection Kit（TK）,and set as the transfection group. The untransfected（empty vector transfected） HT-29 cells were set as the control group. The levels of NGAL mRNA and protein were detected by real-time quantitative PCR（RT-QPCR） and enzyme linked immunosorbent assay（ELISA）respectively. Cell migration,proliferation and apoptosis were examined by wound healing assay,Promega Cell Proliferation Assay G3580 and BD C6 flow cytometry. Results At 72 h after NGAL siRNA transfection,the level of NGAL mRNA in the transfection group was 0. 123 of the control group,which was set as 1. 000（P〈0. 05）. At 48 h after transfection,the level of NGAL protein in the cell culture supernatant of the transfection group was significantly lower than that of the control group [（0. 328 ± 0. 055） μg/L vs（1. 071 ± 0. 143） μg/L,t = 8. 205,P = 0. 001]. At the same time point,the apoptosis rate was also significantly reduced in the transfection group compared with the control group [（18. 87 ± 1. 65） % vs（11. 93 ± 1. 90） %,t =-4. 752,P = 0. 009]. At 24 h after transfection,the migration rate of the transfection group was decreased to only（23. 35 ± 6. 35） % of the control group（t = 11. 930,P〈0. 01）. Moreover,the cell proliferation rate of the transfection group was（90. 00 ± 5. 30） % of the control group（t = 1. 983,P = 0. 118） at 48 h after transfection. Conclusion NGAL siRNA can inhibit the expression of NGAL in colon cancer cells,which subsequently inhibit cell migration and enhance cell apoptosis.

关 键 词：结肠肿瘤/病理学 RNA 小分子干扰 癌基因蛋白质类/代谢 急相蛋白质类/代谢 明胶酶类/代谢 中性白细胞/酶学 基因沉默 细胞增殖 细胞运动 细胞凋亡 

分 类 号：R735.35[医药卫生—肿瘤] R730.23[医药卫生—临床医学]