DNA载体介导的RNAi抑制肝癌细胞的骨桥蛋白表达及侵袭性  

DNA vector-mediated RNAi inhibiting the expression of osteopontin and invasion of human hepatoma cells

作　　者：孙冰生[1] 叶青海[1] 刘道永[1] 任宁[1] 逄锦忠[1] 雷科锋[1] 张巨波[1] 林国领[1] 武金才[1] 李国才[1] 钦伦秀[1] 

机构地区：[1]复旦大学肝癌研究所 中山医院生物医学研究院癌症研究中心,上海200032

出　　处：《中华实验外科杂志》2006年第8期940-942,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(30371378);国家杰出青年科学基金(30325041);教育部跨世纪优秀人才计划;上海市科委科研计划专项基金(03DZ14024)

摘　　要：目的探讨骨桥蛋白(OPN)对高转移潜能人肝癌细胞系HCCLM3侵袭性影响。方法构建两个针对人OPN mRNA干扰质粒pENTR^(TM)/U6-INF(pINF-1、2)及对照质粒pENTR^(TM)/ U6-CTR(pCTR),并将其转染HCCLM3细胞。逆转录-聚合酶链反应(RT-PCR)测定OPN的mR—NA表达量；Western印迹测定OPN蛋白的表达量；噻唑蓝(MTT)比色法实验检测HCCLM3的增殖力；Matrigel侵袭实验检测细胞侵袭力的改变；结果与仅加转染试剂的空白对照组(2．01±0．13)相比。转染24h后,plNF-1组OPN mRNA用RT-PCR无法检测到,pINF-2组mRNA(1．00±0．12)下降50%,而pCTR组mRNA(2．02±0．13)表达差异无统计学意义(P＞0．05)。转染48h后plNF-1组OPN蛋白表达(0．11±0．02)与空白对照组(1．02±0．11)相比下降90%；转染plNF-1后,HCC-LM3细胞的增殖力下降2%。但与对照组相比差异无统计学意义(P＞0．05)。Matrigel侵袭实验结果显示,与空白对照组(28．4±1．5)相比,转染plNF-1后HCCLM3细胞穿过人工基底膜的数量(10．2±0．8)明显减少,差异有统计学意义(P＜0．01)。结论pINF-1可以特异性抑制高转移肝癌细胞系HCCLM3中OPN的表达,并显著降低其侵袭力,但对细胞增殖力影响不大,此技术有望成为抑制肝癌侵袭转移的新途径。Objective To explore the effect of osteoponfin （OPN） on the invasion of hepatoceUular carcinoma cell line HCCLM3. Methods Two double-stranded DNA vectors pENTR^TM/U6-INF （pINF-1,2） targeting the mRNA of human OPN, and the control vector pENTR^TM/U6-CTR （pCTR） mismatching with mRNA of OPN were constructed, and then transfected into human HCCLM3 cells with high metastatic potentials. RT-PCR and Western blotting were used to quantify the mRNA and protein levels of OPN, respectively. The malignant phenotypes of transfected HCCLM3 cells including proliferation and invasive activities were analyzed. Resdts Compared with the OPN mRNA level of control group （2.02 ± 0.13） only transfected with Lipofectamine^TM2000, no mRNA of OPN could be detected 24 h after transfection in the pINF-1 group by RT-PCR and OPN protein level could be decreased by 90 % 48 h after transfection, but 50 % decrease of mRNA in the group of pINF-2 （ 1.00 ± 0.12） and no significant difference could be seen in the group transfected with pCTR （2.02±0.13）.In vitro cellular growth rate was decreased by 2% （P〉0.05） in the group transfected with pINF-1, and migrating number of HCC-LM3 cells （10.2 ± 0.8） transfected with pINF-1 was also significantly decreased （P〈 0.01） as compared with the control group （28.4± 1.5）. Conclusion Sequence-specific shRNA （pINF-1 ） can significantly reduce the OPN expression in HCCLM3 cell line with a high degree of pulmonary metastasis.Suppression of OPN expression in HCCLM3 cells transfected with pINF-1 can induce the decline of invasive ability of HCCLM3, but have no significant effect on the cellular proliferation rate.

关 键 词：骨桥蛋白 RNA干扰 癌 肝细胞 肿瘤 转移 

分 类 号：R735.7[医药卫生—肿瘤] R735.2[医药卫生—临床医学]