Premium
Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines
Author(s) -
Zhang Hejun,
Feng Xiangling,
Liu Weidong,
Jiang Xingjun,
Shan Wenjiao,
Huang Chengan,
Yi Hongmei,
Zhu Bin,
Zhou Wen,
Wang Lei,
Liu Chunmei,
Zhang Lihua,
Jia Wenting,
Huang Wei,
Li Guifei,
Shi Jia,
Wanggou Siyi,
Yao Kaitai,
Ren Caiping
Publication year - 2011
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.23101
Subject(s) - biology , microbiology and biotechnology , methylation , cell culture , carcinogenesis , nasopharyngeal carcinoma , dna methylation , transfection , gene expression , cancer research , loss of heterozygosity , cell growth , gene , genetics , medicine , allele , radiation therapy
Abstract The lactoferrin ( LTF ) gene, located at 3p21.3, behaves like a tumor suppressor gene in diverse tumors. To elucidate the exact role of LTF in NPC, we first detected its expression level in seven NPC cell lines by semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). The results showed the mRNA level of LTF was nearly undetectable in all the seven NPC cell lines, while it could be detected in chronic nasopharyngitis tissues. Subsequently, we used methylation‐specific PCR (MSP), microsatellite assay, PCR‐single‐strand conformation polymorphism (PCR‐SSCP) and sequencing methods to examine the promoter methylation, loss of heterozygosity (LOH) and gene mutation of LTF in NPC cell lines respectively. Consequently, we found that 100% (7 of 7) of NPC cell lines were methylated in LTF promoter, only one cell line (14%, 1 of 7) had LOH and gene mutation of LTF , respectively, while LTF exhibited re‐expression in all cell lines after 5‐aza‐dC treatment, indicating promoter methylation should be the key mechanism causing LTF downregulation in NPC cell lines. Furthermore, patched methylation assay confirmed that promoter methylation could down‐regulate LTF gene expression in NPC cells. Finally, we investigated the function of LTF in NPC cell lines by gene transfection. Restoration of LTF expression in NPC cells resulted in blockage of cell cycle progression, significant inhibition of cell growth and a reduced colony‐formation capacity in vitro and obviously weaker tumor formation potential in vivo. In conclusion, our data indicate LTF may participate in NPC carcinogenesis as a negative effector, that is, a tumor suppressor gene. J. Cell. Biochem. 112: 1832–1843, 2011. © 2011 Wiley‐Liss, Inc.