干预GPC-3基因转录对高转移潜能肝癌细胞增殖的抑制作用  

Intervention of glypican-3 genetic transcription on anti-proliferative effect of hepatoma cells withhigh metastatic potentiality

作　　者：姚敏[1] 王理[1] 时运[1] 钱琦[1] 蔚丹丹[1] 时杨[1] 陆少林[1] 姚登福[1] 

机构地区：[1]医学院南通大学附属医院临床医学研究中心,南通大学226001

出　　处：《中华医学杂志》2014年第32期2544-2548,共5页National Medical Journal of China

基　　金：国家国际科技合作专项（2013DFA32150）;中国博士后科学基金（2013M53139）;江苏省卫生科技项目（K201102）

摘　　要：目的探讨特异性磷脂酰肌醇蛋白多糖3（GPC-3）小发夹RNA（shRNA）转染对高转移潜能肝癌细胞增殖和肝癌生长的抑制作用。方法将构建与筛选GPC．3shRNA最有效序列转染MHCC-97H细胞，以荧光定量聚合酶链反应（PCR）或Western印迹分析mRNA或蛋白表达；以5-乙炔基-2’脱氧尿嘧啶核苷（EdU）、划痕和Transwell试验评估肝癌细胞增殖、迁移和侵袭能力；以Caspase-Glo@3／7荧光法分析细胞凋亡；以裸鼠移植瘤观察对肝癌形成与生长的影响，免疫组化分析瘤组织GPC-3、13-环连蛋白（catenin）、p-GSK3β及细胞周期蛋白（cyclin）D1表达。结果shRNA，质粒转染MHCC-97H细胞效率〉80％，GPC-3mRNA下降75．6％（t=15．473，P〈0．001），蛋白表达受抑；细胞增殖抑制71．1％（t=10．468，P〈0．001）、迁移抑制80．1％（t=32．697，P〈0．001）与侵袭抑制69．1％（t=39．647，P〈0．001）；β-cateninmRNA抑制67．7％（t=18．4，P〈0．001）；GlilmRNA上调53．3％（t=-4．824，P=0．008）；干预组裸鼠肿瘤形成平均11．2d，显著长于阴性组5．5d和空白组5．3d（P〈0．001），瘤体积65．5mm3明显小于空白组404．8mm3和阴性组365．7mm3（P〈0．001）；且瘤组织GPC-3、B-catenin、P-GSK313及cyclinDl等信号分子表达显著下调（P〈0．01）。结论干预GPC-3转录抑制肝癌细胞增殖、迁移和肿瘤生长，提示GPC-3为潜在的肝癌治疗分子靶目标。[ Abstract] Objective To explore the silencing glypican-3 （GPC-3） gene transcription by specific small hairpin RNA （shRNA） on the inhibition of hepatoma cells with high metastatic potentiality and hepatoma growth. Methods After MHCC-97H cells were transfected with higher effective GPC-3-shRNA, GPC-3 mRNA was analyzed by multiple FG-RT-PCR or protein by Western blot. Cell proliferation was detected by 5-ethynyl-2＇-deoxyuridine and sulforhodamine B assay, its migratory metastasis and invasiveness by wound healing or transwell chamber system and cell apoptosis was detected by Caspase-Glo 3/7 Luminescence assay. Nude mice were subcutaneously injected with stable MHCC-97H cells for observing the forming time or volume of xenograft tumors. And the expressions of GPC-3,β-catenin, p-GSK3 β and CyclinD1 were analyzed by immunohistochemistry. Results After shRNA1 transfection with high efficiency （ 〉 80% ）, the expression of GPC-3 was down-regulated to 75.6% （ t = 15. 473, P 〈 0. 001 ） at mRNA level in accordance with its protein, inhibiting cell proliferation （71. lifo, t = 10. 468, P 〈 0. 001 ）notably, decreasing its migration （80. 1%, t = 32. 697, P 〈 0. 001 ） and invasiveness （69. 1%, t = 39. 647, P 〈 0. 001）. 13-catenin was down-regulated （67. 7% , t = 18.4, P 〈0. 001 ） and GliI increased （53.5% , t = - 4. 824, P = 0. 008 ） with its protein. The average forming time of subcutaneous tumors was 1 1. 2 days （d） in the shRNA group and it was siguifieantly longer （P 〈0. 01 ） than that in the control （5.3 d） or shRNA- neg （5.5 d） group. And the average volume （65.5 mm3） of tumors with decreased GPC-3, [3-catenin, p-GSK3β, and cyclinD1 expressions in the shRNA group was significantly smaller （P 〈 0. 01 ） than that in the shRNA-neg （365.7 mm3 ） or control （404. 8 mm3 ） group, respectively. Conclusion Specific shRNAmight intervene effectively the GPC-3 gene transcription and inhibit invasion and tumor growth. Thus GPC-3 may become a potent

关 键 词：癌 肝细胞 磷脂酰肌醇蛋白多糖3 基因沉默 裸鼠 移植瘤 

分 类 号：R735.7[医药卫生—肿瘤]