摘要
目的 探讨小干扰RNA（siRNA）沉默核糖核苷酸还原酶M2（RRM2）基因表达对入骨肉瘤细胞Saos-2生物学影响及分子机制.方法 利用小干扰RNA技术,观察沉默Saos-2细胞中RRM2基因的表达,用实时荧光定量PCR（Real time-PCR）及Western blot技术检测人骨肉瘤细胞Saos-2和人正常成骨细胞hFOB1.19的mRNA及蛋白的表达；用CCK-8方法检测si-RRM2对Saos-2细胞增殖的影响；用Transwell细胞迁移系统检测si-RRM2对Saos-2细胞迁移能力的影响；用酶联免疫吸附试验（ELISA法）检测Saos-2细胞凋亡；采用Western blot技术检测细胞周期调控因子细胞周期素D1（Cyclin D1）和抗凋亡蛋白B细胞淋巴瘤/白血病-2（Bcl-2）蛋白水平变化.结果 Saos-2细胞RRM2 mRNA和蛋白比hFOB1.19细胞高表达；用si-RRM2转染Saos-2细胞后,Real time-PCR结果证实能时间及剂量依赖性下调RRM2基因表达；CCK-8方法结果显示下调RRM2基因会时间及剂量依赖性抑制Saos-2增殖,而人正常成骨细胞增殖抑制没有显著变化；Transwell细胞迁移系统检测显示下调RRM2基因后,显著抑制了Saos-2的迁移能力；si-RRM2联合化疗药物阿霉素促进Saos-2细胞凋亡；Western blot结果显示沉默RRM2后细胞周期调控因子Cyclin D1和抗凋亡蛋白Bcl-2均下调.结论 RRM2的过表达与人骨肉瘤细胞Saos-2的高增殖和迁移能力有关,抑制RRM2的功能是治疗人骨肉瘤的一个潜在的治疗策略.
Objective To investigate the effect of small interference RNA（siRNA） targeting RRM2 on the biological behavior of human osteosarcoma cell line Saos-2 and the molecular mechanisms.Methods RRM2 expression was knocked down in human osteosarcama cell line Saos-2 by RRM2 siRNA.The expression of RRM2 mRNA and protein was determined in human osteosarcoma cell line Saos-2 and human osteoblast-like cell line hFOB1.19 by real time-PCR and Western blot.The cell proliferation was detected by CCK-8.The migration was observed by using transwell system.The apoptotic rate was observed by ELISA.The expression of CyclinD1 and Bcl-2 proteins were detected by Western blot.Results The expression of RRM2 mRNA and protein was higher in Saos-2 than in hFOB1.19.siRRM2 could down-regulate the expression of RRM2 in Saos-2 cells in a time-and concentration-dependent manner.CCK-8 assay showed that si-RRM2 could inhibit the proliferation ability of Saos-2 cells in a time-and concentrationdependent manner,but had no effect on the proliferation of hFOB1.19 cells.Transwell assay indicated that si-RRM2 could inhibit the migration of Saos-2 cells.si-RRM2 combined with adriamycin could increase the apoptosis of Saos-2 cells.Western blot showed that the expression of Cyclin D1 and Bcl-2 were decreased by silencing RRM2.Condusion RRM2 overexpression maybe associate with the osteosarcoma cells proliferation and migration and suppression of its function is a potential therapeutic strategy in osteosarcoma.
出处
《中国基层医药》
CAS
2013年第17期2566-2569,共4页
Chinese Journal of Primary Medicine and Pharmacy
