上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

FKBP12蛋白RNA干扰与回复表达细胞系的建立

陈思，陈鑫，赵永旭，李伟，王毓美   

  1. 上海交通大学医学院/中科院上海生命科学研究院健康科学研究所 干细胞和肿瘤信号转导研究组， 上海 200025
  • 出版日期:2015-03-28 发布日期:2015-03-26
  • 通讯作者: 王毓美, 电子信箱: ymwanganni@yahoo.com。
  • 作者简介:陈思（1988—）， 女， 硕士生; 电子信箱: sichenbio@aliyun.com。
  • 基金资助:

    国家重点基础研究发展计划（“973”计划）(2011CB510105)

Construction of cell lines with shRNA-FKBP12 and stable re-expression of FKBP12

CHEN Si, CHEN Xin, ZHAO Yong-xu, LI Wei, WANG Yu-mei   

  1. Laboratory of Tumor and Stem Cell, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 20025, China
  • Online:2015-03-28 Published:2015-03-26
  • Supported by:

    National Basic Research Program of China, “973”Program, 2011CB510105

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摘要：

目的 建立FKBP12蛋白（FK506 binding-protein 12，基因名FKBP1A）RNA干扰和回复表达的人肺癌细胞系A549，并初步探索FKBP12的功能。方法 设计3条核心序列位于FKBP1A 3′UTR区域的siRNA，它们仅靶向内源性的FKBP12。利用pLKO-1-puro载体包装相应的shRNA慢病毒感染A549，得到FKBP12表达调低的细胞系。使用带neo基因筛选标记的pcDNA3.1载体回复表达FKBP12，并筛选得到不受shRNA干扰的回复表达细胞系。用实时荧光定量PCR和Western blotting检测调低和回复表达效果，并初步分析细胞系表型。结果 FKBP12在mRNA和蛋白质水平上的表达均被抑制，干扰效率在75%以上（P<0.01）。FKBP12不影响A549的细胞形态和增殖效率，而下调mTORC1 （mammalian target of Rapamycin complex 1）下游转录调控相关蛋白4E-BP1 （eIF4E-binding protein 1）的表达。结论 成功构建了FKBP12蛋白敲低和回复表达的A549细胞系，为后续探究FKBP12的功能奠定了基础。

关键词: FKBP12, siRNA, 敲低, mTOR信号通路, A549

Abstract:

Objective To construct cell lines with shRNA-FKBP12 (FK506 binding-protein 12, encoded by the gene FKBP1A) and stable re-expression of FKBP12 in lung cancer cell line A549 and explore the functions of FKBP12. Methods Three siRNAs with core sequences in FKBP1A 3′ UTR region were designed and only targeted endogenous FKBP12. The shFKBP12 in pLKO-1-puro vector was constructed with effective core sequences provided by siRNA. The shFKBP12 was packed in lentivirus and transfected into the A549 cell line, and the cell line with down-regulated expression of FKBP12 was obtained. The expression of FKBP12 was recovered by pcDNA3.1 vector with screening marker of neo gene and the cell line with recovered expression of FKBP12 that was not intervened by shRNA was screened. The outcome of down-regulation of expression and re-expression was evaluated by the real-time PCR and Western blotting and the phenotype of cell line was primary analyzed. Results Both mRNA and protein expressions of FKBP12 were inhibited and the interference efficiency were more than 75% (P<0.01). FKBP12 did not affect the morphology and proliferation of A549 cells, but down-regulated the expression of downstream transcription-regulation protein 4E-BP1 (eIF4E-binding protein 1) of mTORC1 (mammalian target of Rapamycin complex 1). Conclusion A549 cell lines with knockdown and stably re-expression of FKBP12 are successfully constructed which will be helpful for studying the functions of FKBP12.

Key words: FKBP12, siRNA, knockdown, mTOR signaling pathway, A549