基础医学与临床 ›› 2011, Vol. 31 ›› Issue (6): 609-614.

• 研究论文 • 上一篇    下一篇

MicroRNA-29b重组慢病毒表达载体的构建及其抑制胃癌细胞系的迁移和增殖

蒋崇亮1,汪晓艳1,龚佳男2,王芳2,余佳2,冯涛1   

  1. 1. 重庆医科大学基础医学院分子医学与肿瘤研究中心
    2. 中国医学科学院基础医学研究所北京协和医学院基础学院
  • 收稿日期:2011-02-28 修回日期:2011-04-15 出版日期:2011-06-05 发布日期:2011-06-06
  • 通讯作者: 冯涛 E-mail:fengtao9_9@yahoo.com
  • 基金资助:
    国家重点基础研究发展计划

Construction of recombinant lentiviral vector of MicroRNA-29b and its inhibition on the migration and proliferation of gastric cancer cells

Chong-liang JIANG1,Xiao-yan WANG1,Jia-nan GONG2,Fang WANG2,Jia YU3,Tao FENG1   

  1. 1. Molecular Medicine&Cancer Research Center, Institute of Basic Medical Sciences, Chongqing Medical University
    2.
    3. Institute of Basic Medical Sciences, CAMS&PUMC
  • Received:2011-02-28 Revised:2011-04-15 Online:2011-06-05 Published:2011-06-06
  • Contact: Tao FENG E-mail:fengtao9_9@yahoo.com

摘要: 目的 构建has-miR-29b重组慢病毒的表达载体,并观察其对胃癌细胞系迁移和增殖能力的影响。方法 将PCR扩增的miR-29b前体序列和pMIR载体经双酶切后连接产生pMIR-miR-29b慢病毒表达载体。pMIR-miR-29b、Packaging Plasmid(s)和pVSV-G质粒共转染包装细胞系293TN,包装产生慢病毒。使用收获的慢病毒颗粒感染胃癌细胞系HGC-27, 72h后利用real-time PCR检测miR-29b在HGC-27内的表达水平,Western blot检测其靶基因MCL-1的表达。划痕实验和CCK8分别观察在HGC-27细胞中过表达miR-29b后细胞迁移能力及增殖能力的变化。结果 成功构建了miR-29b重组慢病毒的表达载体,感染胃癌细胞HGC-27后,能够成功过表达miR-29b。在HGC-27细胞中过表达miR-29b,其靶基因MCL-1的表达明显被抑制,并且Lenti-29b组与对照组Untreat组和Lenti-vector组相比,Lenti-29b组的迁移能力显著降低(P<0.01);Lenti-29b组与Lenti-vector组相比,Lenti-29b组在48h、72h及96h 3个时间点的增殖能力均显著下降(P<0.01)。结论 成功获得过表达miR-29b的重组慢病毒颗粒,miR-29b能够抑制其靶基因MCL-1的表达,并对HGC-27细胞的迁移和增殖有明显的抑制作用。

关键词: miR-29b, 胃癌细胞, 慢病毒, 细胞迁移, 细胞增殖

Abstract: Objective: To construct the recombinant lentivirus vector expressing hsa-miR-29b and observe the effect of over-expression of miR-29b on gastric cancer cell migration and proliferation. Methods The pMIR-miR-29b lentivirus vector was generated by PCR amplification of miR-29b precursor sequence. 293TN cells were transfected with pMIR-miR-29b、Packaging Plasmid(s) and pVSV-G plasmids to package the recombinant lentivirus. HGC-27 cells were infected with recombinant lentivirus and the expression level of miR-29b was measured by real-time PCR.The expression of target gene MCL-1 was tested by western blot .Scratch test and CCK8 were used to observe the change in cell migration and proliferation after miR-29b was over-expressed in gastric cancer cells. Results The expression vector of 29b restructuring slow virus have been successfully achieved. After infected stomach cancer cell HGC-27, successfully achieved the overexpression of miR-27. The miR-29b that is overexpressed can inhibit the expression of target gene MCL-1 in HGC-27, and inhibit the ability of migration and proliferation of HGC-27. comprised with untreated group and Lenti-vector group, the migration ability of Lenti-29b group is reduced significantly (P<0.01); comprised with Lenti-vector group, the Proliferative capacity of lenti-29b group is reduced significantly at 48h,72h and 96h(P<0.01). Conclusion Restructuring lentivirus particles that has over expression of miR-29b have been successful achieved. MiR-29b can inhibit the expression of target gene MCL-1 and the migration and proliferation of HGC-27.

Key words: miR-29b, Gastric cancer cells, Lentivirus, Cell migration

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