Int J Med Sci 2013; 10(5):498-507. doi:10.7150/ijms.5560 This issue
1. Central Laboratory of Yong-chuan hospital, Chongqing Medical University, Chongqing 402160, China.
2. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chong-qing Medical University, Chongqing 400016, China.
Promyelocytic leukemia (PML) is a cell-growth suppressor, and PML-retinoic acid receptor α (PML-RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Studies have reported that neutrophil elastase(NE) cleaved bcr-1-derived PML-RARα in early myeloid cells leading to the removal of nuclear localization signal (NLS) from PML. The resultant PML without NLS named PML(NLS-). PML(NLS-) mainly locates and functions in the cytoplasm. PML(NLS-) may act in different ways from PML, but its biological characteristics have not been reported. In this study, the PML (NLS-) was silenced with shRNA [HL-60/pPML(NLS-)-shRNA] and over-expressed by preparation of recombinant adenovirus [HL-60/pAd-PML(NLS-)]. The mRNA and protein expression of PML(NLS-) were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect apoptotic cells. The transcription of BCL-2, BAX and C-MYC was detected in HL-60/pAd-PML(NLS-) cells. Our results showed that compared to the control group, the expression of PML(NLS-) was significantly reduced in the HL-60/pPML(NLS-)-shRNA cells, and increased significantly in the HL-60/pAd-PML(NLS-) cells. The proliferation was significantly inhibited in the HL-60/pPML(NLS-)-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-PML(NLS-) cells treated with 60 μmol/L emodin. FCM revealed the apoptosis increased in HL-60/pPML(NLS-)-shRNA cells, and decreased in the HL-60/pAd-PML(NLS-) cells. The expression of BAX decreased significantly, while that of BCL-2 and C-MYC increased significantly in HL-60/ pAd-PML(NLS-) cells. Down-regulation of PML(NLS-) expression inhibits the proliferation and induces the apoptosis of HL-60 cells. On the contrary, over-expression of PML(NLS-) promotes the proliferation and reduce the emodin-induced apoptosis of HL-60 cells.
Keywords: PML(NLS-), shRNA, over-expression, proliferation, apoptosis.