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