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中国癌症防治杂志

中国癌症防治杂志 ›› 2013, Vol. 5 ›› Issue (1): 6-11.doi: 10.3969/j.issn.1674-5671.2013.01.02

• 基础研究 • 上一篇    下一篇

miR-21真核表达载体的构建及其对胃癌细胞增殖与凋亡的影响

  

  1. 南京医科大学附属常州市第二人民医院病理科；南京医科大学附属常州市第二人民医院肿瘤中心实验室
  • 出版日期:2013-03-25 发布日期:2013-04-07
  • 基金资助:

    常州市卫生局青年人才科研基金资助项目（NQ 201104）

Construction of a recombinant eukaryotic expression vector of miR-21 and its effects on proliferation and apoptosis in gastric cancer cells

  • Online:2013-03-25 Published:2013-04-07
  • Contact: 孙阳阳 E-mail:sunyangyang527111@163.com

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被引次数

6

摘要： 目的 探讨人微小RNA-21（miR-21）重组表达质粒对人胃癌SGC-7901细胞增殖与凋亡的影响及可能机制。方法 设计合成靶向miR-21的互补双链DNA，退火后与真核表达载体pPG-miR-EGFP克隆连接，构建pPG-miR-21-EGFP重组表达质粒，利用脂质体LipofectamineTM2000将鉴定正确的重组表达质粒pPG-miR-21-EGFP转染人胃癌SGC-7901细胞，荧光显微镜下观察转染效率。实验分为3组：空白对照组、空载体组、重组表达质粒组。以RT-PCR检测转染前后胃癌细胞miR-21的表达水平。MTT法评价重组表达质粒抑制肿瘤细胞生长的效果，流式细胞术及Hoechst 33258法检测转染后胃癌SGC-7901细胞周期的分布和凋亡。Western blot法、免疫细胞化学法分别检测转染前后PTEN、PCNA、PDCD4、bcl-2、cyclin D1 蛋白表达的水平。结果 酶切及测序鉴定证实成功构建重组质粒pPG-miR-21-EGFP，转染人胃癌SGC-7901细胞后，荧光显微镜下观察转染效率达75%。RT-PCR检测显示重组表达质粒组胃癌细胞miR-21的表达下调，与空白对照组和空载体组比较，差异具有统计学意义（P<0.05）。Western blot法、免疫细胞化学法检测结果显示重组表达质粒组胃癌细胞cyclin D1、PCNA、bcl-2蛋白的表达下调（P<0.05），而PDCD4、PTEN蛋白的表达上调（P<0.05）。重组表达质粒组的细胞生长缓慢，凋亡指数增加。流式细胞术检测结果显示重组表达质粒组G1期细胞的百分比较空白对照组增加［（69.71±0.314）% vs （50.1±0.331）%］，S期细胞较空白对照组明显减少［（14.68±0.448）% vs （28.47±0.316）%］ （P<0.05）。结论 重组表达质粒pPG-miR-21-EGFP可显著下调miR-21在胃癌SGC-7901细胞中的表达，并在一定程度上抑制胃癌细胞的增殖，促进其凋亡，使较多的细胞停留在G1期，S期细胞减少，其作用机制可能是通过下调cyclin D1、PCNA、bcl-2蛋白的表达，上调PDCD4、PTEN蛋白的表达而实现的。

关键词: 胃肿瘤, 微小RAN-21, 增殖, 凋亡

Abstract: Objective To construct a recombinant expression plasmid encoding human miR-21，and explore the effects of miR-21 expression on the proliferation and apoptosis of gastric cancer cells. Methods Double-stranded DNA encoding human miR-21 was designed and used to construct eukaryotic expression plasmid pPG-miR-EGFP.The plasmid was transfected into the human gastric cancer cell line SGC-7901 using LipofectamineTM2000，and the transfection rate was monitored by fluorescence microscopy. Levels of miR-21 were analyzed by RT-PCR in three sets of cells：untransfected cells（control group），cells transfected with empty vector（empty group）， and cells transfected with expression plasmid（expression group）.In addition，cell growth and proliferation were assayed using MTT，and cell cycle progression and apoptosis were analyzed using flow cytometry based on Hoechst 33258 staining.Expression of PTEN，PCNA， PDCD4，bcl-2，and cyclin D1 were analyzed using Western blotting and immunohistochemistry. Results The recombinant expression plasmid pPG-miR-21-EGFP was successfully constructed and transfected into 75% of cells. Levels of miR-21 were significantly lower in the expression group than in the empty or control groups（t=7.12，5.78，P<0.05）.Expression of cyclin D1，PCNA，and bcl-2 was significantly lower in the expression group than in the other two groups（P<0.05），while expression of PDCD4 and PTEN was significantly higher（P<0.05）.Cells in the expression group showed lower growth and higher apoptosis rates than cells in the control group（P<0.05），and flow cytometry revealed a greater proportion of cells in G1 phase［（69.71±0.314）% vs （50.1±0.331）%］ and a smaller proportion in S phase［（14.68±0.448）% vs （28.47±0.316）%］（P<0.05 in both cases）. Conclusions Recombinant expression plasmid pPG-miR-21-EGFP can down-regulate miR-21 expression in human gastric cancer SGC-7901 cells and suppress their proliferation to some extent.It can also promote apoptosis，such that many cells stop at the G1 phase and do not proceed to S phase.These effects on growth and apoptosis may be related to down-regulation of cyclin D1，PCNA，and bcl-2 and/or with up-regulation of PDCD4 and PTEN.

Key words: Gastric neoplasms, miR-21, Proliferation, Apoptosis