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D_3受体的表达及功能  

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

作　　者：张晔[1] 朱晓燕[2] 刘宇健[1] 卢建[1] 

机构地区：[1]第二军医大学基础医学部病理生理学教研室,上海200433 [2]基础医学部生理学教研室

出　　处：《第二军医大学学报》2006年第10期1076-1080,共5页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(30270522).~~

摘　　要：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D3[1,25(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。Objeetive：To construct an expression vector containing RNA interfering （RNAi） sequence targeting human vitamin D3 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）2D3-induced phosphorylation of PKC. Cell counting and MTT were used to evaluate their influence on the inhibitory effects of 1,25（OH）2 D3 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）2 D3 on the cell proliferation, and in quick phosphorylation of PKC induced by 1,25 （OH）2 D3 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）2D3 mediated by VDR, which paves a way for further study on the biological functions of VDR and vitamin D3.

关 键 词：RNA干扰 骨肉瘤 受体 维生素D3 

分 类 号：R738.1[医药卫生—肿瘤]