shRNA靶向阻断NF-κB p65对人肝癌细胞迁移增殖能力的抑制作用及机制  

Silencing NF-κB p65 by shRNA dramatically suppressed migration and proliferation of human hepatocellular carcinoma cells through downregulating IL-1βexpression

作　　者：闫奔 付晓燕 马超 崔瑞翔 彭美玉 梁淑娟 YAN Ben;FU Xiao-yan;MA Chao;CUI Rui-xiang;PENG Mei-yu;LIANG Shu-juan(Key Lab for Immunology in Universities of Shandong Province,School of Basic Medical Sciences,Shandong Second Medical University,Weifang,Shandong 261053,China)

机构地区：[1]山东第二医科大学基础医学院,山东省高校免疫学重点实验室,山东潍坊261053

出　　处：《环境与健康杂志》2023年第4期292-299,共8页Journal of Environment and Health

基　　金：国家自然科学基金(81972695);山东省医药卫生科技发展计划(2018WS065)

摘　　要：目的 分析shRNA阻断NF-κB p65对人肝癌细胞(HepG2细胞)迁移增殖能力的影响及机制。方法 通过在线软件设计4段人NF-κB p65的shRNA序列(shRNA1至shRNA4)及一段非沉默对照序列,经过EcoR I和Age I双酶切后克隆至pLKO.1-sp6-pgk-GFP慢病毒表达载体。在HEK-293T细胞中包装制备慢病毒,浓缩10倍后,感染人HepG2细胞。采用免疫荧光(IF)法和流式细胞(FACS)技术检测慢病毒感染效率,采用免疫蛋白印迹(Western blot)法检测NF-κB p65水平。采用酶联免疫吸附法(ELISA法)测定IL-1水平。采用划痕实验、Transwell细胞迁移实验检测细胞迁移能力。采用CCK8细胞计数法和14天克隆形成实验分析细胞的增殖能力。结果 获得了4种表达靶向人NF-κB p65的shRNA慢病毒和1种对照病毒。各类shRNA慢病毒感染HepG2细胞的效率基本一致。WB结果证实4种shRNA对NF-κB p65表达都有干扰效果,其中,shRNA1和shRNA4的抑制效果最好。在划痕实验和小室迁移实验中,与感染了非沉默对照序列慢病毒的HepG2细胞相比,感染了NF-κB p65 shRNA1和shRNA4慢病毒的HepG2细胞的迁移能力和增殖能力显著降低(P<0.01),同时其克隆形成能力也明显低于非沉默对照序列组(P<0.01)。利用LPS诱导实验证实,与非沉默对照序列组感染的HepG2细胞相比,感染shRNA1和shRNA4慢病毒的HepG2细胞分泌的IL-1的水平显著降低(P<0.01)。与感染shRNA1和shRNA4慢病毒的HepG2细胞相比,外源性补充IL-1后,感染前述慢病毒的HepG2细胞增殖和迁移能力显著升高。结论 shRNA阻断HepG2细胞中NF-κB p65表达后能够进一步下调IL-1分泌,进而抑制人肝癌细胞的迁移和增殖能力。Objective To elucidate the effect and mechanisms of shRNA targeting NF-κB p65 on migration and proliferation of human hepatocellular carcinoma cells.Methods Four specific shRNA sequences targeting human NF-κB p65(shRNA1 to shRNA4)and one non-silencing control sequence(Ctrl)were designed.These sequences were cloned into lentiviral plasmid pLKO.1-sp6-pgk-GFP with EcoRI and AgeI restrictive enzymes.Lentiviruses expressing each shRNA or Ctrl sequence were concentrated 10 times through ultracentrifugation.Expression efficiency of lentivirus in human hepatocellular carcinoma cell line HepG2 cells was analyzed through immunofluorence(IF)and FACS using GFP as an indicator.The level of NF-κB p65 protein was detected by western blotting(WB).Migration of HepG2 cells was assessed through scratching assay and chamber based transwell assay.Proliferation activity was calculated by CCK8 assay and a 14-day colony formation assay.Secreted IL-1βin supernatant of HepG2 cells treated with LPS was detected by ELISA.Results shRNA1 to shRNA4 expressing lentiviruses against human NF-κB p65 protein and the non-silencing Ctrl lentivirus attained equal infection efficiency in HepG2 cell when assessed through either IF or FACS assay.In the scratch test and cell migration test,the migration and proliferation ability of HepG2 cells infected with NF-κB p65 shRNA1 and shRNA4 lentiviruses were significantly reduced compared with HepG2 cells infected with non-silent control sequence lentivirus(P<0.01),and its cloning ability was also significantly lower than that of the non-silent control sequence group(P<0.01).LPS induction experiments confirmed that the level of IL-1 secreted by HepG2 cells infected with shRNA1 and shRNA4 lentivirus was significantly reduced compared with HepG2 cells infected with non-silent control sequence group(P<0.01).Compared with HepG2 cells infected with shRNA1 and shRNA4 lentiviruses,after exogenous supplementation with IL-1,the proliferation and migration ability of HepG2 cells infected with the aforementioned len

关 键 词：NF-κB p65 肝癌细胞 迁移 增殖 IL-1 

分 类 号：R994.6[医药卫生—毒理学]