shRNA腺病毒介导的JNK1 RNAi抑制U87MG人胶质瘤细胞的增殖

JNK1 Knockdown via Adenovirus-mediated shRNA Inhibited Cell Proliferation in U87MG Gliobalstoma Cells

目的研究腺病毒介导的shRNA对U87MG人胶质瘤细胞JNK1基因表达的抑制作用,以及对细胞增殖的影响。方法设计合成针对JNK1基因以及无义对照的shRNA序列,通过腺病毒介导shRNA的表达来下调JNK1的表达;Q-PCR和Western blot验证病毒效果;用BrdU掺入的方法来检测细胞的增殖情况。结果限制性酶切及DNA测序结果证实shRNA插入片段完全正确;Q-PCR和Western blot结果表明JNK1的表达与对照组相比明显下降;BrdU掺入结果表明Ad-shJNK1处理组细胞增殖显著降低。结论重组的JNK1shRNA腺病毒构建成功,JNK1的下调明显抑制了细胞的增殖,为深入研究JNK信号通路在神经肿瘤中的作用奠定基础。

Objective To investigate the effects of adenovirus-mediated shRNA targeting c-jun N-terminal kinase 1（JNK1） in U87MG human glioblastoma cells,so as to reveal the relationship between JNK1 and cell proliferation in brain tumor.Methods shRNA sequence against JNK1 and nonsense sense oligonucleotides were designed;after producing recombinant adenovirus,Q-PCR and Western blot were used to test knockdown effect in U87MG cells;BrdU incorporation was used to observe cell proliferation.Results Recombinant vector was confirmed by restriction endonuclease analysis and DNA sequencing;Q-PCR and Western blot results indicated the expression of JNK1 was significantly decreased compared with the other control groups.BrdU incorporation revealed cell proliferation rate was markedly inhibited in JNK1 RNAi group in comparison with control groups.Conclusion Recombinant adenovirus expressing shRNA targeting JNK1 was constructed successfully,and cell proliferation was significantly inhibited after JNK1 knockdown in U87MG cells;it paves the way for further research on JNK pathway involving in development of brain tumor.