转染沉默IGF1R基因的肝癌细胞株增殖、迁移及侵袭能力观察
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摘要
目的设计并筛选高效沉默胰岛素样生长因子1受体(IGF1R)基因的小干扰RNA(siRNA),构建其慢病毒表达载体,观察转染沉默IGF1R基因的肝癌细胞株增殖、迁移及侵袭能力。方法根据siRNA设计原则,参照IGF1R mRNA序列设计3对siRNA序列及1对阴性对照序列,转染肝癌细胞株Huh7 24 h后采用实时荧光定量PCR检测IGF1R mRNA,筛选干扰效率最高的siRNA序列。构建该序列的慢病毒表达载体,转染293T细胞进行病毒包装。将包装好的慢病毒表达载体感染肝癌细胞株Huh7和Hep3B,筛选沉默IGF1R基因表达的稳定细胞株。将上述稳定细胞株扩大培养,观察细胞IGF1R mRNA表达变化及细胞增殖、迁移与侵袭能力变化。结果实时荧光定量PCR显示,IGF1R_002序列在100 nmol/L浓度时抑制效率最高,达78.6%。与未感染任何病毒的Huh7、Hep3B细胞,感染p LVX-shRNA2空载体的Huh7、Hep38细胞相比,感染携带IGF1R干扰序列慢病毒的Huh7、Hep3B细胞IGF1R mRNA表达水平显著下调,细胞增殖活性明显降低,细胞迁移和侵袭能力明显受到抑制(P均<0.01)。结论筛选出1对高效沉默IGF1R基因的siRNA序列,即IGF1R_002;该siRNA介导的IGF1R基因沉默可明显抑制肝癌细胞株Huh7和Hep3B增殖、迁移与侵袭。
Objective To design and screen an effective small interfering RNA( siRNA) targeting human insulin-like growth factor 1 receptor( IGF1R) gene,to construct the siRNA lentiviral vector,and to observe its effect on the proliferation,migration and invasion of hepatocellular carcinoma cells. Methods According to siRNA design principle,three siRNAs targeting IGF1 R gene and one negative control siRNA were designed and synthesized. They were transfected into Huh7 cells. IGF1 R mRNA expression levels in Huh7 cells were detected by real-time fluorescent quantitative PCR at 24 h after transfection,and we screened the most effective siRNA. After lentiviral expression vector was constructed,it was transfected into 293 T cells and packed into lentiviral. Huh7 and Hep3 B cells were infected with the p LVX-shRNA2-IGF1R_002lentiviral to screen the stable cell lines. The stable cell lines were cultured in large. We detected the changes in the IGF1 R mRNA expression,cell proliferation,cell migration and invasion. Results The real-time fluorescent quantitative PCR showed that IGF1R_002 showed the highest inhibition effect( relative inhibition rate 78. 6%) at the concentration of 100 nmol / L. The expression levels of IGF1 R mRNA in Huh7 and Hep3 B cells with the lentiviral were significantly reduced,the proliferation abilities were inhibited,and the abilities of cell migration and invasion were significantly decreased as compared with those of Huh7 and Hep3 B cells without any virus infection and Huh7 and Hep3 B cells infected with p LVX-shRNA2 empty vector( all P < 0. 01). Conclusions The most effective siRNA targeting human IGF1 R gene,namely IGF1R_002,is screened out. The most effective siRNA-mediated IGF1 R gene silencing can significantly suppress the proliferation,migration and invasion of Huh7 and Hep3 B cells.
Objective To design and screen an effective small interfering RNA( siRNA) targeting human insulin-like growth factor 1 receptor( IGF1R) gene,to construct the siRNA lentiviral vector,and to observe its effect on the proliferation,migration and invasion of hepatocellular carcinoma cells. Methods According to siRNA design principle,three siRNAs targeting IGF1 R gene and one negative control siRNA were designed and synthesized. They were transfected into Huh7 cells. IGF1 R mRNA expression levels in Huh7 cells were detected by real-time fluorescent quantitative PCR at 24 h after transfection,and we screened the most effective siRNA. After lentiviral expression vector was constructed,it was transfected into 293 T cells and packed into lentiviral. Huh7 and Hep3 B cells were infected with the p LVX-shRNA2-IGF1R_002lentiviral to screen the stable cell lines. The stable cell lines were cultured in large. We detected the changes in the IGF1 R mRNA expression,cell proliferation,cell migration and invasion. Results The real-time fluorescent quantitative PCR showed that IGF1R_002 showed the highest inhibition effect( relative inhibition rate 78. 6%) at the concentration of 100 nmol / L. The expression levels of IGF1 R mRNA in Huh7 and Hep3 B cells with the lentiviral were significantly reduced,the proliferation abilities were inhibited,and the abilities of cell migration and invasion were significantly decreased as compared with those of Huh7 and Hep3 B cells without any virus infection and Huh7 and Hep3 B cells infected with p LVX-shRNA2 empty vector( all P < 0. 01). Conclusions The most effective siRNA targeting human IGF1 R gene,namely IGF1R_002,is screened out. The most effective siRNA-mediated IGF1 R gene silencing can significantly suppress the proliferation,migration and invasion of Huh7 and Hep3 B cells.
引文
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[2]Habib R,Akhtar J,Taqi M,et al.Lentiviral vector-mediated survivin shRNA delivery in gastric cancer cell lines significantly inhibits cell proliferation and tumor growth[J].Oncol Rep,2015,34(2):859-867.
[3]Weng CJ,Hsieh YH,Tsai CM,et al.Relationship of insulin-like growth factors system gene polymorphisms with the susceptibility and pathological development of hepatocellular carcinoma[J].Ann Surg Oncol,2010,17(7):1808-1815.
[4]Singh P,Alex JM,Bast F.Insulin receptor(IR)and insulin-like growth factor receptor 1(IGF-1R)signaling systems:novel treatment strategies for cancer[J].Med Oncol,2014,31(1):805.
[5]Adachi Y,Yamamoto H,Ohashi H,et al.A candidate targeting molecule of insulin-like growth factor-I receptor for gastrointestinal cancers[J].World J Gastroenterol,2010,16(46):5779-5789.
[6]汤绍辉,吴胜兰,王旷靖,等.h AFP及h TERT双启动子调控的针对人IGF-Ⅱ基因的siRNA特异性抑制人肝癌细胞生长[J].中国病理生理杂志,2013,29(8):1422-1427.
[7]Wu XY,Wu ZF,Cao QH,et al.Insulin-like growth factor receptor-1 overexpression is associated with poor response of rectal cancers to radiotherapy[J].World J Gastroenterol,2014,20(43):16268-16274.
[8]Aleem E,Nehrbass D,Klimek F,et al.Upregulation of the insulin receptor and typeⅠinsulin-like growth factor receptor are early events in hepatocarcinogenesis[J].Toxicol Pathol,2011,39(3):524-543.
[9]Nussbaum T,Samarin J,Ehemann V,et al.Autocrine insulin-like growth factor-Ⅱstimulation of tumor cell migration is a progression step in human hepatocarcinogenesis[J].Hepatology,2008,48(1):146-156.
[10]Alexia C,Bras M,Fallot G,et al.Pleiotropic effects of PI-3'kinase/Akt signaling in human hepatoma cell proliferation and druginduced apoptosis[J].Ann N Y Acad Sci,2006,1090:1-17.
[11]Yan XD,Yao M,Wang L,et al.Overexpression of insulin-like growth factor-I receptor as a pertinent biomarker for hepatocytes malignant transformation[J].World J Gastroenterol,2013,19(36):6084-6092.