中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2016, Vol. 45 ›› Issue (7): 604–609.doi: 10.12007/j.issn.0258-4646.2016.07.007

• 论著 • 上一篇    下一篇

RNA 干扰下调 MBP-1 对骨肉瘤 Saos-2 细胞生物学的影响

施鑫鹤 1,耿晢 2,施星臣 3,马克君 1,朱宏文 1,任雯 1,周雅丽 1   

  1. 1 兰州大学第二医院中心实验室,兰州 730030;2 兰州大学基础医学院生物化学及分子生物学研究所,兰州 730000;3 兰州第二人民 医院骨科,兰州 730030
  • 收稿日期:2015-12-07 出版日期:2016-07-29 发布日期:2016-06-27
  • 作者简介:施鑫鹤(1973 -),男,副主任检验师,硕士 .
  • 基金资助:
    甘肃省自然科学研究基金计划一般项目(1308RJZA152)

Effects of RNA Interfering of MBP-1 on Proliferation of Saos-2 Cell Line

SHI Xinhe1,GENG Zhe2,SHI Xingchen3,MA Kejun1,ZHU Hongwen1,REN Wen1,ZHOU Yali1   

  1. 1 Central laboratory of Lanzhou University Second Hospital,Lanzhou University,Lanzhou 730030,China;2 Institute of Biochemistry and Molecular Biology, Lanzhou Medical College,Lanzhou University,Lanzhou 730000,China;3 Department of Bone Surgery,The Second People’s Hospital of Lanzhou,Lanzhou 730030, China
  • Received:2015-12-07 Online:2016-07-29 Published:2016-06-27

摘要: 目的 探讨 c-myc 启动子结合蛋白 1(MBP-1)基因沉默对人骨肉瘤 Saos-2 细胞生长的影响。方法 实验分为 3 组:正常 对照组(未转染骨肉瘤细胞)、阴性对照组(转染错义序列组)和沉默组(转染 MBP-1 shRNA 组)。设计 2 条 MBP-1 基因的 RNA 干 扰片段及 1 条阴性对照 siRNA,并与 pSIREN-retroQ 质粒连接。将重组 pSIREN-retroQ 质粒通过 Lipofectamine 2000 脂质体转染骨 肉瘤 Saos-2 细胞。实时 PCR 和 Western blot 分别检测 MBP-1 表达。CCK-8 法对 MBP-1 沉默后骨肉瘤 Saos-2 细胞生长进行检测。 Western blot 检测对照组和沉默组 cyclin D1 和 cyclin E 的表达。结果 通过 PCR 扩增及测序,说明已成功构建 MBP-1 沉默及对 照 重 组 pSIREN - retroQ 质 粒 。 实 时 PCR 结 果 显 示 ,沉 默 组 MBP - 1 mRNA 相 对 表 达 量 与 对 照 组 相 比 显 著 下 调(P < 0.05)。 Western blot 结果显示,沉默组 MBP-1 蛋白表达量与对照组相比也显著下调。CCK-8 法结果表明,沉默组细胞在 48、72 和 96 h 时 增殖能力均比对照组显著升高(P < 0.05)。沉默组 cyclin D1 和 cyclin E 的表达显著高于对照组(P < 0.05)。结论 MBP-1 基因 沉默后骨肉瘤 Saos-2 细胞生长被明显促进,为寻找骨肉瘤基因治疗新靶点打下基础。

关键词: 骨肉瘤 Saos-2 细胞, RNA 干扰, c-myc 启动子结合蛋白 1, 细胞生长

Abstract: Objective To investigate the effects of c-myc promoter binding protein 1(MBP-1)gene on the proliferation of human Saos-2 osteosarcoma cells in vitro. Methods Saos-2 cells were divided into three groups:blank control group(untransfected cells),negative group(cells transfected with missense sequence)and experimental group(cells transfected with MBP-1 shRNA). Two MBP-1 shRNA sequences and one negative control shRNA sequence were designed,synthesized and cloned into pSIREN-retroQ plasma. Then the recombinant plasmids were constructed and transfected into human Saos-2 osteosarcoma cells by Lipofectamine 2000. The expressions of MBP-1 mRNA and protein in Saos-2 cells were detected by real-time PCR and Western blot,respectively. The effects of altered expression of MBP-1 on cell proliferation were measured by CCK-8 cell proliferation assay. The expressions of cyclin D1 and cyclin E in Saos-2 were determined by Western blot. Results PCR and sequencing results indicated that the recombinant plasmids pSIREN-retroQ was constructed. The relative expression level of MBP-1 mRNA in the MBP-1 siRNA transfection group was significantly decreased than that in blank control group(P < 0.05). Compared with the blank control group,the expression levels of MBP-1 protein in the experimental group also significantly decreased. The proliferation abilities of Saos-2 cells at 48,72,and 96 hours after MBP-1 siRNA transfection were significantly increased than those in the blank control group(P < 0.05). Compared with the blank control group,the expression levels of cyclin D1 and cyclin E protein in the experimental group also significantly increased(P < 0.05). Conclusion Knockdown of the expression of MBP-1 gene promotes the proliferation of human Saos-2 osteosarcoma cells. MBP-1 gene may become the new target of gene therapy for osteosarcoma.

Key words: osteosarcoma Saos-2 cells, RNA interfering, c-myc promoter binding protein 1, cell proliferation

中图分类号: 

  • R738.3
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