摘要
癌胚抗原(CEA)作为肿瘤标志物,在多种恶性肿瘤中高表达.为探索CEA在肿瘤发生发展过程中的生物学作用,本研究以结肠癌细胞株8307为研究对象,应用RNAi技术抑制CEA在8307细胞中的表达,观察其对8307细胞生长的影响.依据siRNA设计原则,设计合成靶向CEA的shRNA并克隆到pSilencer2.1-U6 neo载体中,成功构建了CEA shRNA表达载体,通过脂质体介导转染8307细胞,经G418筛选出抑制CEA表达的稳定细胞.RT-PCR、Western印迹分别检测CEA mRNA及蛋白表达水平,MTT及集落形成实验检测细胞增殖活性.实验结果显示,构建的CEA shRNA表达载体明显抑制CEA mRNA及蛋白水平的表达.MTT实验中,CEA表达下调的8307细胞生长活性降低,与对照组相比,抑制率达39%(P<0.05),细胞集落形成能力也显著降低(P<0.05).上述结果初步证明,构建的CEA shRNA表达载体能明显降低CEA的表达,并抑制结肠癌细胞增殖活性.
Carcino-embryonic antigen （CEA）, as a tumor marker, is up-regulated in many types of human cancers. RNA interference （RNAi） technology was applied to silence CEA in colorectal carcinoma cell line 8307 and the effects on cell growth was studied. CEA-targeting shRNA was designed and cloned into the pSilencer2, l-U6 neo vector, then transfected into 8307cells with lipofectamine and selected with G418 for stable clones. The CEA expression was examined by RT-PCR and Western blotting. The cell proliferation was measured by MTT and colony formation assays. Our resuhs showed that the expression of CEA-shRNA significantly reduced, the 8307 cell proliferation was inhibited by 39 % compared with the control group, and the colony formation was also inhibited （ P 〈 0.05 ）. The study indicated that reducing CEA expression with shRNA could inhibit the proliferation of colorectal carcinoma cells.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2009年第1期78-82,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目(No.30873017)~~
关键词
癌胚抗原
RNA干扰
SHRNA
结肠癌
carcino-embryonic antigen（CEA）
RNAi
shRNA
colorectal carcinoma
