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目的构建核蛋白1-短发夹RNA(NUPR1-sh RNA)慢病毒载体定向敲低NUPR1基因,研究NUPR1基因下调对人多发性骨髓瘤U266细胞增殖及凋亡的影响。方法以正常浆细胞为对照,通过实时荧光定量PCR(q RT-PCR)检测骨髓瘤U266细胞和RPMI8226细胞的NUPR1 m RNA水平。设计针对NUPR1基因的sh RNA并包装慢病毒载体感染U266细胞,流式细胞术观察转染效率,q RT-PCR、Western blot法验证NUPR1基因干扰效果,CCK-8法及台盼(trypan)蓝染色检测细胞增殖,流式细胞术及Hoechst染色观察细胞凋亡。结果与正常人浆细胞相比,U266、RPMI8226细胞NUPR1 m RNA水平较高;U266细胞慢病毒感染效率达80%,NUPR1-sh RNA慢病毒载体构建成功。敲低NUPR1水平后,U266细胞增殖减弱,抑制率高达(58.71±1.64)%;与阴性对照组和空白对照组相比,敲低NUPR1水平后,U266细胞凋亡率明显增加。结论敲低U266细胞NUPR1水平后,可明显抑制U266细胞增殖并促进其凋亡。
Objective To construct NUPR1-sh RNA lentivirus vector and knock down NUPR1 gene to investigate the effect of NUPR1 gene downregulation on the proliferation and apoptosis of human multiple myeloma U266 cells. Methods Normal plasma cells were used as control. The levels of NUPR1 mRNA in myeloma U266 cells and RPMI8226 cells were detected by real-time fluorescence quantitative PCR (q RT-PCR). The sh RNA targeting NUPR1 gene was designed and packaged into lentiviral vector to infect U266 cells. The transfection efficiency was observed by flow cytometry. The interference effect of NUPR1 gene was confirmed by q RT-PCR and Western blot. The CCK-8 and trypan blue Cell proliferation, flow cytometry and Hoechst staining were used to observe apoptosis. Results Compared with normal human plasma cells, the level of NUPR1 mRNA in U266 and RPMI8226 cells was higher. The lentivirus infection rate was 80% in U266 cells and the NUPR1-sh RNA lentiviral vector was constructed successfully. After knocking down the level of NUPR1, the proliferation of U266 cells was weakened and the inhibition rate was as high as (58.71 ± 1.64)%. Compared with the negative control group and the blank control group, the apoptosis rate of U266 cells was significantly increased after knockdown of NUPR1. Conclusion Knockdown of NUPR1 in U266 cells can significantly inhibit the proliferation and promote the apoptosis of U266 cells.