靶向siRNA阻断NF-κB信号通路对神经母细胞瘤侵袭转移的影响  

Effects on invasion and metastasis of neuroblastoma by targeted siRNA blockage of nf-kappa B pathway

作　　者：傅春燕[1] 董蒨[1] 张虹[1] 鹿洪亭[1] 郝希伟[1] 关鸽[1] 周泉[2] 

机构地区：[1]青岛大学附属医院小儿外科,266003 [2]青岛大学附属医院中心实验室,266003

出　　处：《中华小儿外科杂志》2014年第2期115-120,共6页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金面上项目（81272986）

摘　　要：目的探讨重组慢病毒介导siRNA（小干扰RNA）靶向阻断NF-κB信号通路对人神经母细胞瘤QDDQ-NM1细胞侵袭转移的影响。方法根据人p65基因序列，设计针对基因不同靶点的siRNA1、siRNA2、siRNA3、siRNA44条siRNA序列及其对照序列NC，慢病毒介导转染QDDQ-NM1，采用实时荧光定量PCR（RT-PCR）和Westernblot法从设计的4条干扰序列中筛选出siRNAl序列在转染72h干扰效果最好；在转染72h后，采用RTPCR和Westernblot法检测NF-κB下游基因CXCR4、VEGnrFmRNA转录和蛋白表达情况；Transwell小室检测各组间瘤细胞的转移侵袭能力。结果转染72h后，PT-PCR显示，siRNAl-NF-κBCXCR4、VEGFmRNA表达分别为空白对照组0．66倍、0．76倍，差异有统计学意义（P〈0．05）；Westernblot显示，siRNA1-NF-κBCXCR4、VEGF蛋白表达分别为空白对照组0．67倍、0．76倍，差异有统计学意义（P〈0．05）；Transwell小室显示，Ⅰ（实验组）、Ⅱ（空载对照组）、Ⅲ（空白对照组）穿过Matrigel胶的细胞数分别为34．6000±3．9749；45．6000±2．3021；54．8000±2．3874，各组间差异有统计学意义（P〈0．01）。结论慢病毒介导的siRNA通过下调NF-κB下游基因CXCR4、VEGF的表达抑制神经母细胞瘤的侵袭转移，可为下一步肿瘤治疗提供理论基础。Objective To explore the effects of NF-kappa B pathway blocked by small interfer- ing RNA （siRNA） mediated by lentivirus on the invasion and metastasis of human neuroblastoma cancer cell QDDQNM1. Methods According to human p65 gene sequences, 4 siRNAs targeting dif- ferent targets of p65 and NC control sequences were synthesized and transfected into QDDQ-NM1. And the best sequence of transfection interference effect was screened by real-time fluorescent quanti- tative reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The interfering effect of siRNA1 was optimal among 4 siRNAs after 72 h. At this time, the expressions of NF-κB downstream genes CXCR4 and VEGF at mRNA and protein levels were also detected. The cellular in- vasion and metastasis capability of different groups were evaluated by Transwell assay. Results At 72 h post-transfeetion, compared with control groups, RT-PCR showed that the expressions of CXCR4 and VEGF mRNA targeting siRNA1 respectively decreased （0. 66 times, 0. 76 times, P〈0. 05）. And Western blot indicated that the expression of CXCR4 and VEGF protein respectively decreased （0. 67 times, 0. 76 times, P〈0. 05） ; Transwell demonstrated that the number of cells passing through Ma- trigel rubber of siRNA1 interference, transfection control and control groups were 34. 6000 ± 3. 9749, 45. 6000 ± 2. 3021 and 54. 8000 ± 2. 3874 respectively. Statistical significance existed among different groups （P〈0. 01）. Conclusions NF-κB p65 gene silencing by siRNA inhibits the invasion and metas- tasis of human neuroblastoma cancer cell QDDQ-NM. Thus it may provide theoretical basis for pro- spective cancer therapy.

关 键 词：神经母细胞瘤 RNA干扰 慢病毒感染 NF-ΚB 受体 CXCR4 血管内皮生长因子 

分 类 号：R739.41[医药卫生—肿瘤]