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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (2) : 148-152     DOI: 10.1007/s11684-009-0032-7
RESEARCH ARTICLE |
Cloning of human XAF1 gene promoter and assay of its transcription activity in a variety of cell lines
Qiong CHEN, Qing YU, Yuhu SONG, Peiyuan Li, Ying CHANG, Zhijun WANG, Lifeng LIU, Wei WU, Jusheng LIN()
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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Abstract  

To investigate the regulation of tumor suppressor XAF1 gene expression in digestive system cancers, we studied XAF1 gene promoter transcription activity and mRNA level in digestive system cancer cell lines (human hepatoma cell line HepG2, human colon cancer cell line LoVo, and human gastric cancer cell line AGS) and nontumor cell lines (human embryonic liver cell line L02 (L02 cells) and human embryonic kidney 293 cells [HEK293 cells]) as controls. 1395-bp-promoter fragment of XAF1 gene was amplified by polymerase chain reaction (PCR) and cloned into pGL3-basic vector and pEGFP-1 vector to assay its promoter transcription activity. The plasmids were transfected into a variety of cell lines by lipofectamine 2000. The promoter transcription activity was determined by dual-luciferase report assay, and enhanced green fluorescent protein (EGFP)-positive cells were detected by fluorescence microscope. The expression of XAF1 mRNA in HEK293 and L02 were significantly higher than that in any of the three digestive system cancer cell lines. The dual-luciferase reporter assay showed that the promoter transcription activity in digestive system tumor cell lines transfected with pGL3-XAF1p promoter was apparently lower than that of both HEK293 and L02 cells. Expression of green fluorescent protein (GFP) under the control of XAF1 promoter in the three digestive system cancer cell lines was lower than that of both HEK293 and L02 cells. The activities of pGL3-XAF1p in the three digestive system cancer cell lines after treatment with heat stress were significantly lower than those in the unstressed cells. The results suggested that remarkably down-regulated XAF1 mRNA expression in digestive system cancer cell lines may be due to loss of transcription activity of XAF1 promoter.

Keywords gene      X-linked inhibitor of apoptosis protein associated factor-1 (XAF1)      promoter      transcription regulation     
Corresponding Authors: LIN Jusheng,Email:jslin@tjh.tjmu.edu.cn   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0032-7     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I2/148
Fig.1  1395-bp fragment of XAF1 gene promoter amplified by PCR. M: marker DL2000; 1, 2, 3: 1395-bp fragment of XAF1 gene promoter.
Fig.2  Agarose gel electrophoresis identification of (a) pGL3-XAF1p and (b) pEGFP-XAF1p. M: DNA marker 3; 1: pGL3-XAF1p; 2: pGL3-XAF1p double digested by I and I; 3: pEGFP-XAF1p; and 4: pEGFP-XAF1p double digested by I and H I.
Fig.3  The activity of pGL3-XAF1p in a variety of cell lines. 293: human embryonic kidney 293 cells; L02: human embryonic liver cell line L02 cells; HepG2: human hepatoma cell line HepG2; AGS: human gastric cancer cell line AGS; and LoVo: human colon cancer cell line LoVo.
Fig.4  The activity of pGL3-XAF1p in the cancer cell lines. HepG2: human hepatoma cell line HepG2; AGS: human gastric cancer cell line AGS; LoVo: human colon cancer cell line LoVo.
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