干扰IGF-1R基因表达对肝癌细胞裸鼠移植瘤生长影响研究  

Down-regulating insulin-like growth factor-1 receptor gene transcription on effect of hepatoma growth inhibition

作　　者：姚敏[1,2] 严晓娣[1] 王理[3] 王司晔[1] 邱历伟[1] 姚登福[1] 

机构地区：[1]南通大学附属医院临床医学研究中心,江苏南通226001 [2]南通大学医学院免疫学教研室,江苏南通226001 [3]南通大学医学院医学信息学教研室,江苏南通226001

出　　处：《中华肿瘤防治杂志》2015年第19期1528-1533,共6页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家国际科技合作项目(2013DFA32150);南通市科技项目(HS2014078)

摘　　要：目的分析人肝癌胰岛素样生长因子-1受体(insulin-like growth factor-1receptor,IGF-1R)表达并观察干预其基因转录对裸鼠肝癌移植瘤的抑制作用。方法以免疫组化法分析肝癌组织IGF-1R表达;构建IGF-1R-shRNA真核表达质粒并筛选最有效序列;转染至肝癌PLC/PRF/5细胞,CCK-8法分析细胞增殖变化;经G418筛选稳定株,接种裸鼠皮下成瘤;依肿瘤体积绘制生长曲线,并经HE染色证实。以荧光定量PCR技术(FQ-RT-PCR)分析IGF-1R mRNA改变,以蛋白质印迹法分析IGF-1R蛋白水平。结果肝癌组IGF-1R阳性率为82.5%,癌周组为42.5%,非癌组为10%;IGF-1R强度组间差异有统计学意义,K-W法检测Hc=51.51,P<0.001。低分化组肝癌细胞中IGF-1R阳性表达率为100.0%(6/6),显著高于高分化肝癌组37.5%(3/8),P<0.05。人肝LO2细胞中IGF-1R未见明显表达,3种肝癌细胞株中均不同程度的表达,以PLC/PRF/5细胞表达量最高。FQ-RT-PCR检测显示,IGF-1R mRNA表达受抑,且以shRNA4效果最佳,抑制率为(59.6±2.8)%(n=3);蛋白质印迹法检测结果显示,IGF-1R蛋白水平相一致,平均抑制率为(58.3±9.4)%(n=3)。CCK-8法分析细胞增殖,干扰组细胞较阴性对照组均明显抑制,72h时抑制率为(63.87±3.9)%,t=19.244,P<0.001;在mRNA水平平均抑制率(59.6±2.8)%,呈时间依赖性,增殖周期发生G1期阻滞;shRNA4干扰PLC/PRF/5细胞IGF-1R表达,促进肝癌细胞凋亡,干扰组肝癌细胞凋亡率为44.84%,显著高于空白组的6.62%(P<0.001)及阴性对照组的4.02%(P<0.001)。裸鼠移植瘤证实,对照组和阴性对照组IGF-1R过表达,分别为7.2±1.1和6.8±1.1,差异无统计学意义,t=0.577,P>0.05;干预组IGF-1R下调为1.8±0.5,显著低于对照组(t=11.184,P<0.01)及阴性对照组(t=9.450,P<0.01)。结论干预IGF-1R基因转录有效抑制裸鼠移植瘤生长,提示IGF-1R有望成为肝癌基因治疗的有效靶点。OBJECTIVE To investigate the expression of human hepatoma insulin-like growth factor-1 receptor （IGF-1R） and observe silencing its gene transcription on effects of nude mice xenograft growth suppression. METHODS The IGF-1R expressions in human hepatoma tissues were analyzed by immunohisto chemistry. Pairs of IGF-1R shRNAs were designed and synthesized according to IGF-1R sequence, screened the most effective one and transfected into PLC/ PRF/5 cells. Cell proliferations were analyzed by CCK-8 assay, and transplanted into nude mice to establish xenograft with monitored the tumor growth daily, and morphological alteration was confirmed by H^E staining. The alterations of IGF1R mRNA or IGF-1R level were analyzed by FQ-RT-PCR or Western Blotting, respectively. RESULTS The inci- dence of IGF-1R was 82.5% in human hepatoma tissues and 42.5% in their surrounding and 10% in noncancerous tissues with significantly difference （K-W method, Hc= 51.51, P〈0.01） and 100.0% （6/6） in the poorly differentiated group which was remarkable higher （P〈0.05） than that in the high differentiated group （37.5 %, 3/8）. Except of LO2 cells, the different expression of IGF-1R was observed among hepatoma PLC/PRF/5, HepG2, and Bel-7404 cells, with the highest level in PLC/PRF/5 cells. After screening, the IGF-1R-shRNA4 was the most efficiency for interfering IGF-1R gene transcription with the inhibiting rate E （59- 6± 2.8）%, n = 3] at IGF-1R mRNA level by FQ-RT-PCR assay, and alteration of IGF-1R level in line with mRNA with average inhibiting rate [（58.3 ± 9.4）%, n= 3]confirmed by Western Blotting. The cell proliferation in the shRNA group were significantly suppressed [（63.87±3.9）%, t= 19. 244,P〈0. 0011 at 72 h analyzed by the CCK-8 assay and IGF-1R mRNA inhibition （59.6±2.8）% in a time-dependent manner, with the cell cycles arrested in G1 phase and increasing cell apoptosis was 44. 84% （P〈0. 001） which was more than that in the control （6.62%） or neg-shRNA group （4. 02%）. The nude m

关 键 词：肝肿瘤 癌 肝细胞 IGF一1R 基因沉默 移植瘤 免疫组织化学 

分 类 号：R735.7[医药卫生—肿瘤]