利用载体介导的RNA干扰技术抑制人骨肉瘤细胞HOS-8603内维生素D3受体的表达及功能

目的:构建由载体介导的抑制人维生素D3受体(VDR)表达的RNA干扰载体,并在人骨肉瘤细胞中初步研究其对VDR表达及功能的影响.方法:通过在线筛选设计了两段针对人VDR的RNAi序列,并与载体pSilencer-2.1-U6连接,获得了表达载体pSilencer-2.1-U6-VDR1与pSilencer-2.1-U6-VDR2.利用脂质体转染法分别转入人骨肉瘤细胞系HOS-8603细胞中,通过Western 印迹法检测其对内源性VDR蛋白表达、对1,25-二羟维生素D31,25(OH)2D3]激活PKC作用的影响,利用细胞计数和MTT的方法检测其对1,25(OH)2D3抑制HOS-8603细胞增殖作用的影响.结果:在分别转染pSilencer-2.1-U6-VDR1/2的HOS-8603细胞中,与对照细胞比较,内源性VDR蛋白的表达、1,25(OH)2D3对细胞的增殖抑制作用以及1,25(OH)2D3快速诱导PKC磷酸化的作用均有显著降低.结论:构建的表达载体pSilencer 2.1-U6-VDR1与pSilencer 2.1-U6-VDR2均能在细胞水平显著抑制内源性VDR基因的表达,并能有效阻断一些由VDR介导的1,25(OH)2D3生物学作用,为进一步研究VDR与维生素D3的功能提供了一个有用工具.

Inhibitory effect of vector-mediated RNAi technique on expression and function of vitamin D_3 receptor in human osteosarcoma cell line HOS-8603

Objective:To construct an expression vector containing RNA interfering(RNAi) sequence targeting human vitamin D_3 receptor(VDR) protein gene,and to investigate its inhibitory effect on the expression and function of VDR in a human osteosarcoma cell line.Methods: Two small interfering RNA sequences targeting VDR were designed by online screening and were cloned into the expression vector(pSilencer-2.1-U6).Then the products,pSilencer-2.1-U6-VDR1 and pSilencer-2.1-U6-VDR2,were separately transfected into a human osteosarcoma cell line(HOS-8603) by liposome.Western blot was employed to determine their effect on VDR protein expression and 1,25(OH)_2D_3-induced phosphorylation of PKC.Cell counting and MTT were used to evaluate their influence on the inhibitory effects of 1,25(OH)_2D_3 on HOS-8603 cells proliferation.Results: Compared to HOS-8603 cells transfected with control vectors,those transfected with pSilencer2.1-U6-VDR1 or pSilencer2.1-U6-VDR2 had significant decreases in the expression of VDR protein, in the inhibitory effects of 1,25(OH)_2D_3 on the cell proliferation,and in quick phosphorylation of PKC induced by 1,25(OH)_2D_3.Conclusion: The vectors constructed in the present study(pSilencer2.1-U6-VDR1 and pSilencer2.1-U6-VDR2) can significantly suppress the VDR expression in HOS-8603 cells,and block some biological activities of 1,25(OH)_2D_3 mediated by VDR, which paves a way for further study on the biological functions of VDR and vitamin D_3.