CRISPR-Cas9 mediated CD133 knockout inhibits colon cancer invasion through reduced epithelial-mesenchymal transition

We previously reported that CD133, as a putative cancer stem cell marker, plays an important role in cell proliferation and invasion in colon cancer. To understand the role of CD133 expression in colon cancer, we evaluated the inhibitory effect of CD133 in colon cancer cells. In this study, we generated CD133knockout colon cancer cells (LoVo) using the CRISPR-Cas9 gene editing system. CD133+ colon cancer cells (LoVo) were infected with the lentiviral vector carrying CD133 gRNA and purified cell by culturing single cell colonies. CD133knockout cells was validated by western blot and flow cytometry analysis. In functional study, we observed a significant reduction in cell proliferation and colony formation in CRISPR-Cas9 mediated CD133 knockout cells in compare with control (P &lt; 0.001). We also found the anticancer effect of stattic was dependent on CD133 expression in colon cancer cells. Although CD133knockout cells could not completely block the tumorigenic property, they showed rema...此前我们已有报道称，CD133作为一种推定的癌症干细胞标志物（cancer stem cell marker），在结肠癌的细胞增殖与侵袭过程中发挥重要作用。为阐明CD133表达在结肠癌中的功能，我们评估了敲除CD133对结肠癌细胞的抑制效应。本研究中，我们借助CRISPR-Cas9基因编辑系统构建了CD133基因敲除的结肠癌细胞（LoVo细胞）。我们将携带CD133向导RNA（gRNA）的慢病毒载体感染CD133阳性的结肠癌细胞（LoVo），并通过单细胞菌落培养的方式纯化得到目标细胞。我们通过蛋白质印迹（western blot）与流式细胞术（flow cytometry）分析验证了CD133基因敲除细胞的构建有效性。功能实验结果显示，与对照组相比，CRISPR-Cas9介导的CD133基因敲除细胞的增殖能力与集落形成能力均显著降低（P < 0.001）。我们还发现，斯塔替克（stattic）的抗癌活性在结肠癌细胞中依赖于CD133的表达。尽管CD133基因敲除细胞无法完全阻断其致瘤特性，但它们表现出……