Zeng Chensi, Yi Bei, Li Xingxin, Chen Jianbin
Department of Hematology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Department of Hematology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Sep;33(9):1240-1246.
Objective To construct nuclear protein 1-short hairpin RNA (NUPR1-shRNA)-expressing lentiviral vector targeting NUPR1 gene, and investigate the effect of NUPR1 gene silencing on the proliferation and apoptosis of human multiple myeloma U266 cells. Methods The normal plasma cells were used as controls and the mRNA expression level of NUPR1 in myeloma cell lines U266 and RPMI8226 were detected by quantitative real-time PCR (qRT-PCR). Then the shRNA plasmid targeting NUPR1 gene was constructed and transfected into U266 cells. The transfection efficiency was detected by flow cytometry. The interference effect of NUPR1 gene was detected by qRT-PCR and Western blotting. The cell proliferation was analyzed by CCK-8 assay and trypan blue viability counting. The cell apoptosis was determined by flow cytometry and Hoechst staining. Results The mRNA expression level of NUPR1 in U266 and RPMI8226 were higher than that in the normal plasma cells. NUPR1-shRNA-expressing lentiviral vector was successfully constructed, and flow cytometry shows that the transfection efficiency was above 80%. Compared with the negative control group and the blank control group, the proliferation of U266 cells was significantly inhibited with the inhibitory rate being (58.71±1.64)%, and the apoptosis rate of U266 cells was significantly elevated in NUPR1 knockdown group. Conclusion Down-regulating the expression of NUPR1 in U266 cells can inhibit cell proliferation and promote its apoptosis.
目的 构建靶向核蛋白1（NUPR1）基因的短发夹RNA（shRNA）表达慢病毒载体，探讨NUPR1基因沉默对人多发性骨髓瘤U266细胞增殖和凋亡的影响。方法 以正常浆细胞为对照，采用实时荧光定量PCR（qRT-PCR）检测骨髓瘤细胞系U266和RPMI8226中NUPR1的mRNA表达水平。构建靶向NUPR1基因的shRNA质粒并转染U266细胞，通过流式细胞术检测转染效率。采用qRT-PCR和蛋白质免疫印迹法检测NUPR1基因的干扰效果。采用CCK-8法和台盼蓝活细胞计数分析细胞增殖情况。采用流式细胞术和Hoechst染色检测细胞凋亡。结果 U266和RPMI8226中NUPR1的mRNA表达水平高于正常浆细胞。成功构建了表达NUPR1-shRNA的慢病毒载体，流式细胞术显示转染效率在80%以上。与阴性对照组和空白对照组相比，NUPR1基因敲低组U266细胞增殖明显受抑制，抑制率为（58.71±1.64）%，细胞凋亡率明显升高。结论 下调U266细胞中NUPR1的表达可抑制细胞增殖并促进其凋亡。
