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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.
Carcinoembryonic antigen (CEA) as a tumor marker is highly expressed in a variety of malignant tumors.In order to explore the biological role of CEA in tumorigenesis, colon cancer cell line 8307 was used as research object, and RNAi technique was used to inhibit CEA In 8307 cells and observe its effect on the growth of 8307. According to the principle of siRNA design, the shRNA targeting CEA was designed and synthesized and cloned into pSilencer2.1-U6 neo vector, and the CEA shRNA expression vector was successfully constructed. The expression of CEA mRNA and protein was detected by RT-PCR and Western blot respectively, and the cell proliferation activity was detected by MTT assay and colony formation assay.Experimental results showed that the constructed cells Of CEA shRNA expression vector significantly inhibited the expression of CEA mRNA and protein.MTT experiment, CEA expression decreased 8307 cell growth inhibition, compared with the control group, the inhibition rate was 39% (P <0.05), colony formation ability (P <0.05) .The above results preliminarily proved that the constructed CEA shRNA expression vector can significantly reduce the expression of CEA and inhibit the proliferation activity of colon cancer cells.