International Journal of Medical Sciences

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16 December 2017

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Int J Med Sci 2014; 11(3):247-254. doi:10.7150/ijms.6518

Research Paper

NLS-RARα Promotes Proliferation and Inhibits Differentiation in HL-60 Cells

Xiu-Xiu Hu 1,2, Liang Zhong2, Xi Zhang 2, Yuan-Mei Gao 2, Bei-Zhong Liu 1,2✉

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, Chongqing Medical University, Chongqing 400016, China.

Abstract

A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα 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 the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The expression of C-MYC increased strikingly in HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. Down-regulation of NLS-RARα expression inhibited the proliferation and induced the differentiation of HL-60 cells. On the contrary, over-expression of NLS-RARα promoted proliferation and reduced the ATRA-induced differentiation of HL-60 cells.

Keywords: NLS-RARα, shRNA, over-expression, HL-60 cell, proliferation, differentiation

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How to cite this article:
Hu XX, Zhong L, Zhang X, Gao YM, Liu BZ. NLS-RARα Promotes Proliferation and Inhibits Differentiation in HL-60 Cells. Int J Med Sci 2014; 11(3):247-254. doi:10.7150/ijms.6518. Available from http://www.medsci.org/v11p0247.htm