miR-21慢病毒干扰载体的构建及其对胆管癌细胞增殖和迁移能力的影响

Construction of lentiviral vector knocking down miR-21 and its effects on proliferation and migration of cholangiocarcinoma cell

目的构建miR-21慢病毒干扰载体,并观察其对胆管癌细胞增殖和迁移能力的影响。方法针对目标序列,设计2条合适的PCR引物进行退火得到插入片段序列。将慢病毒干扰载体双酶切,纯化后与退火产物进行定向连接,产生慢病毒干扰质粒和阴性对照质粒,将其与慢病毒包装质粒混合物共转染293T,包装产生慢病毒。使用收获的慢病毒感染胆管癌细胞HUCCT1,荧光显微镜观察感染效率,细胞增殖和划痕实验分别检测HUCCT1下调miR-21后细胞增殖能力和迁移能力的改变。结果成功构建了miR-21慢病毒干扰载体。感染胆管癌细胞HUCCT1后,miR-21表达降低;下调miR-21(LV-anti-miR21)组和感染空白慢病毒载体(LV-GFP)组相比,LV-anti-miR21组增殖能力和迁移能力均明显下降(P<0.05)。结论成功构建miR-21慢病毒干扰载体;下调miR-21能够抑制胆管癌细胞的增殖,降低胆管癌细胞的迁移能力。

Objective To constrcuct a lentiviral vector knocking down miR-21 and observe the effects of knocking down miR-21 on the proliferation and migration of cholangiocarcinoma cells.Methods To get the inserted fragment,2 suitable PCR primers were annealed targeting the sequence.The LV-anti-miR21 and LV-GFP vector were generated by the connection of the purified lentiviral vector and annealing product.The 293T cells were cotransfected with the lentiviral vector and the lentiviral-packaged plasmids to package the recombinant lentivirus.HUCCT1 cholangiocarcinoma cells were infected with recombinant lentivirus.The infection efficiency was observed by fluorescence microscopy.The scratch test and cell proliferation assays were used to detect the changes in cell migration and proliferation after miR-21 was knocked down.Results The lentiviral vector knocking down miR-21 was successfully constructed.HUCCT1 cholangiocarcinoma cells expressed lower miR-21 after infected by the recombinant lentivirus.Compared with the LV-GFP group,the proliferation and migration of LV-anti-miR21 group was significantly reduced（P0.05）.ConclusionThe recombinant lentivirus knocking down miR-21 has been constructed successfully.Knocking down miR-21 can inhibit the proliferation and invasiveness of cholangiocarcinoma cells.