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p LKO.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pc DNA3.1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m TORC1(mammalian target of Rapamycin complex 1)下游转录调控相关蛋白4E-BP1(e IF4E-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。
Objective To construct cell lines with shRNA-FKBP12 （FK506 binding-protein 12, encoded by the gene FKBPIA） 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 FI（BPIA 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.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2015年第3期301-307,共7页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家重点基础研究发展计划("973"计划)(2011CB510105)~~
