Yi Hong-mei, Li Yan-chun, Zhong Ren-hua
Department of Pathology, Hunan Province People's Hospital, Changsha 410005, China.
Zhonghua Zhong Liu Za Zhi. 2010 Oct;32(10):729-33.
To investigate the expression of LTF mRNA in several nasopharyngeal cancer (NPC) cell lines, and analyze the relationship between the genetic and epigenetic changes and expression of LTF gene.
The expression level of LTF was detected in NPC cell lines HNE1, HNE2, HNE3, CNE1, CNE2, 5-8F, 6-10B cells and tissues of 15 cases of chronic nasopharyngitis by RT-PCR. The LTF protein level was analyzed by Western blotting in 6-10B cells. Then LOH, mutation and methylation status of LTF was examined by microsatellites analysis, PCR-SSCP, MSP and bisulfite genomic sequencing, respectively.
15 chronic nasopharyngitis tissues showed stable LTF expression, while there were weak expression in 6-10B cells and absent expression in remaining detected NPC cell lines. There was a significantly lower LTF expression in chronic nasopharyngitis tissues (Z = -3.738, P = 0.000). No LTF protein expression was observed in 6-10B cells. LOH analysis demonstrated that allele loss of LTF wasn't found in NPC cell lines. LTF mutation was noted in 14.3% (1/7) of NPC cell lines. DNA sequencing confirmed the mutation point in the promoter region (-305 bp to -50 bp) was at -218 bp (del T) of LTF gene in the HNE1 cell line. Methylation of LTF gene was not found in chronic nasopharyngitis. However, methylation of LTF promoter was detected in all NPC cell lines. LTF mRNA expression was increased in 5-8F and 6-10B cell lines after treatment with 5-aza-2-deoxycytidine.
There is an inactivation of expression of LTF gene in the NPC cell lines. Its molecular mechanism may be related with methylation of promoter region and deletion mutation.
研究乳铁传递蛋白（LTF）mRNA在几种鼻咽癌（NPC）细胞系中的表达情况，并分析LTF基因的遗传和表观遗传变化与表达之间的关系。
采用逆转录-聚合酶链反应（RT-PCR）检测NPC细胞系HNE1、HNE2、HNE3、CNE1、CNE2、5-8F、6-10B细胞以及15例慢性鼻咽炎组织中LTF的表达水平。通过蛋白质免疫印迹法（Western blotting）分析6-10B细胞中LTF蛋白水平。然后分别采用微卫星分析、聚合酶链反应-单链构象多态性分析（PCR-SSCP）、甲基化特异性PCR（MSP）和亚硫酸氢盐基因组测序检测LTF的杂合性缺失（LOH）、突变和甲基化状态。
15例慢性鼻咽炎组织中LTF表达稳定，而6-10B细胞中LTF表达较弱，其余检测的NPC细胞系中均未表达。慢性鼻咽炎组织中LTF表达明显较低（Z = -3.738，P = 0.000）。在6-10B细胞中未观察到LTF蛋白表达。LOH分析表明，NPC细胞系中未发现LTF等位基因缺失。14.3%（1/7）的NPC细胞系中检测到LTF突变。DNA测序证实，HNE1细胞系中LTF基因启动子区域（-305 bp至-50 bp）的突变点位于-218 bp（del T）处。慢性鼻咽炎中未发现LTF基因甲基化。然而，在所有NPC细胞系中均检测到LTF启动子甲基化。用5-氮杂-2'-脱氧胞苷处理后，5-8F和6-10B细胞系中LTF mRNA表达增加。
NPC细胞系中LTF基因表达失活。其分子机制可能与启动子区域甲基化和缺失突变有关。
