[Over-expression of neutrophil elastase promotes proliferation and inhibits apoptosis in K562 cells].

ABSTRACT

OBJECTIVE: To establish recombinant adenovirus carrying human neutrophil elastase (NE) gene using AdEasy system, over-express NE in K562 cell line and observe the effects of NE on K562 cell proliferation and apoptosis. METHODS: NE gene was amplified with RNA extracted from acute premyelocytic leukemia (APL) HL-60 cells as a template using reverse transcription-PCR. The coding sequence was cloned into shuttle plasmid pAdTrack-CMV to obtain the recombinant plasmid named pAd-NE. After digested with HindIII and EcoRV and sequenced, the pAd-NE was transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-NE was digested with PacI and transfected into AD293 cells for packaging. Fourteen days later, primary recombinant adenovirus Ad-NE was harvested, and then subjected to five cycles of amplification, titer determination and PCR identification. K562 cells were infected by the recombinant adenovirus. The infection efficiency was observed under a fluorescence microscope and detected by flow cytometry. Western blotting was used to detect NE expression. The proliferation of K562 cells was detected by CCK-8 assay. Cell cycle and apoptosis was measured by annexin V/PI accompanied by flow cytometry. RESULTS: HindIII and EcoRV digestion and sequencing suggested that the recombinant vector Ad-NE was successfully constructed. The recombinant plasmid Ad-NE was packaged in AD293 cells as expected. Following five-cycle amplification, the viral titer was up to 1.64 × 10¹² pfu/mL. GFP expression observed by fluorescence microscopy and flow cytometry implied that the infection efficiency of Ad-NE in K562 cells reached about 80%. Western blotting showed that NE expression was up-regulated in K562 cells. CCK-8 assay revealed that the proliferation of K562 cells over-expressing NE was enhanced. Meanwhile, flow cytometry indicated that the K562 cells were arrested in S phase and the apoptosis rate was highly reduced. CONCLUSION: Over-expressed NE in K562 leukemia cells could promote cell proliferation, inhibit apoptosis and block cell cycle in S phase.