pcDNA3.1-CD74-ROS1重组质粒构建与表达对肺癌细胞增殖及迁移的影响

Construction of pcDNA3.1-CD74-ROS1 Recombinant Plasmid and Effect of CD74-ROS1 Expression on Proliferation and Migration of Lung Cancer Cells

目的构建C-ros致癌基因1(ROS1)融合基因CD74-ROS1重组质粒,并转染A549细胞株,检测其对肺癌细胞迁移能力的影响。方法通过PCR技术获得CD74和ROS1基因片段,利用融合PCR法构建CD74-ROS1融合基因,构建pc DNA3.1-CD74-ROS1重组质粒,通过脂质体转染技术转入A549细胞中,利用Real-time PCR和Western blot技术检测CD74-ROS1的表达。MTT法观察细胞增殖情况,结晶紫染色法观察细胞形态变化,划痕实验检测CD74-ROS1对肺癌细胞迁移能力的影响。结果酶切及测序结果表明CD74-ROS1融合基因构建成功,阳性克隆的CD74-ROS1蛋白表达显著增加。CD74-ROS1过表达导致细胞增殖能力增强,细胞形态上发生上皮-间质样转变且细胞迁移能力增强。结论pc DNA3.1-CD74-ROS1重组质粒构建成功,并在A549细胞中稳定表达,CD74-ROS1过表达能够促进肺癌细胞增殖、形态转变且增强其迁移能力。

Objective To construct the recombinant plasmid carrying CD74-ROS1 fusion gene, and explore its effect on migration ability of A549 cells. Methods CD74 and ROS1 genes were obtained through PCR. The CD74-ROS1 fusion gene was constructed by fusion PCR. We inserted the fusion gene into the eukaryotic expression vector pc DNA3.1, and transfected the pc DNA3.1-CD74-ROS1 recombinant plasmid into A549 cells. The expression of CD74-ROS1 was detected by real-time PCR or Western blot. The cell proliferation was observed by MTT method. The cell morphological or cell migration ability were tested by crystal violet staining or wound healing assay. Results The CD74-ROS1 fusion gene was identified by double enzyme digestion and DNA sequencing. The expression of CD74-ROS1 protein was significantly increased after transfection. The overexpression of CD74-ROS1 enhanced the cell proliferation, changed the cell morphology and increased the ability of cell migration. Conclusion pc DNA3.1-CD74-ROS1 recombinant plasmid is successfully constructed and expressed stably in A549 cells. CD74-ROS1 overexpression could promote cell proliferation and the morphological changes and enhance the migration ability of lung cancer cells.