[Over-expression of prohibitin gene promotes apoptosis in retinoic acid-resistant acute promyelocytic leukemia cell line NB4-R1].

OBJECTIVE: To construct a eukaryotic expression vector carrying human prohibitin gene and study the effect of prohibitin over-expression on the apoptosis of retinoic acid-resistant acute promyelocytic leukemia NB4-R1 cells.

METHODS: The prohibitin gene was amplified by PCR, and then cloned into pEGFP-N1-3FLAG eukaryotic expression vector. Positive clones were selected by PCR screening and identified by DNA sequencing. The positive recombinant vector pEGFP-N1-3FLAG-prohibitin was transferred into NB4-R1 cells. The over-expression of prohibitin was verified by real-time PCR and Western blotting, respectively. The apoptosis rate of the NB4-R1 cells transfected with pEGFP-N1-3FLAG-prohibitin was measured by flow cytometry combined with annexin V-FITC/PI staining.

RESULTS: PCR screening and DNA sequencing demonstrated that the recombinant plasmid of pEGFP-N1-3FLAG-prohibitin was constructed successfully; and the transfection efficiency of NB4-R1 cells reached more than 70%. Moreover, qRT-PCR showed that the expression of prohibitin mRNA of transfection group (OE) increased by (1.64±0.37) times and (1.58±0.43) times (P<0.05) compared with blank control group (CON) and negative control group (NC), respectively. Western blotting showed that the expression of prohibitin protein of OE group increased by (1.91±0.33) times and (1.99±0.37) times (P<0.05) compared with CON and NC, respectively; Flow cytometry indicated that the NB4-R1 cell apoptosis rates in CON, NC and OE groups were respectively (5.88± 0.43)%, (6.63±0.46)% and (28.22±1.33)%, and the apoptosis rate of OE group was elevated by (3.80±0.24) times and 3.39± 0.30 times compared with CON and NC(P<0.05), respectively.

CONCLUSION: The up-regulated expression of prohibitin could promote NB4-R1 cell apoptosis.