Laboratory of Cellular and Developmental Biology, Life Science College, North-east Agricultural University, Harbin, 150030, China.
Cell Biol Int. 2018 Sep;42(9):1132-1140. doi: 10.1002/cbin.10979. Epub 2018 Jul 23.
Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle-derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual-luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU-positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P-AKT. The expression of P-Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3-kinase). In contrast, repression of COL8A1 reduced the number of EdU-positive cells and expression of CCNB1 and P-AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual-luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.
VIII 型胶原α 1 链（COL8A1）是细胞外基质的组成部分。我们之前的研究表明，COL8A1 与肌肉源性卫星细胞（MDSCs）的增殖有关。此外，已经证明 COL8A1 促进平滑肌细胞和肝癌细胞的增殖。因此，我们预测 COL8A1 与牛 MDSCs 的增殖有关，牛 MDSCs 在研究中有潜在的应用价值。在这项研究中，我们使用 CRISPR/Cas9 技术构建了激活和抑制牛 MDSCs 中 COL8A1 的载体，并通过 EdU 标记、Western blot 和双荧光素酶报告基因检测来确定 COL8A1 调节的影响。结果表明，COL8A1 的激活增加了 EdU 阳性细胞的数量和增殖标志物细胞周期蛋白 B1（CCNB1）和 P-AKT 的表达。在添加 LY294002（一种能够阻断磷酸肌醇 3-激酶信号转导通路的蛋白激酶抑制剂）后，P-Akt 的表达没有变化。相反，COL8A1 的抑制减少了 EdU 阳性细胞的数量和 CCNB1 和 P-AKT 的表达。我们还观察到 EGR1 的过表达和抑制分别导致 COL8A1 的上调和下调。双荧光素酶报告基因检测显示，EGR1 调节 COL8A1 启动子的活性。据我们所知，这是第一项研究表明 EGR1 正向调节 COL8A1 的表达，COL8A1 进而通过 PI3K/AKT 信号通路促进牛 MDSCs 的增殖。
