title: Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines.
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.
authors.affiliation: Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Key Laboratory for Carcinogenesis of Chinese Ministry of Health, Changsha, Hunan 410078, China.
authors.forename: Hejun
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiangling
authors.initials: X
authors.lastname: Feng
authors.forename: Weidong
authors.initials: W
authors.lastname: Liu
authors.forename: Xingjun
authors.initials: X
authors.lastname: Jiang
authors.forename: Wenjiao
authors.initials: W
authors.lastname: Shan
authors.forename: Chengan
authors.initials: C
authors.lastname: Huang
authors.forename: Hongmei
authors.initials: H
authors.lastname: Yi
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.forename: Wen
authors.initials: W
authors.lastname: Zhou
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Chunmei
authors.initials: C
authors.lastname: Liu
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Wenting
authors.initials: W
authors.lastname: Jia
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Guifei
authors.initials: G
authors.lastname: Li
authors.forename: Jia
authors.initials: J
authors.lastname: Shi
authors.forename: Siyi
authors.initials: S
authors.lastname: Wanggou
authors.forename: Kaitai
authors.initials: K
authors.lastname: Yao
authors.forename: Caiping
authors.initials: C
authors.lastname: Ren
mesh_terms: D000818:Animals; D001483:Base Sequence; D002277:Carcinoma; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D005260:Female; D016193:G1 Phase; D006801:Humans; D007781:Lactoferrin / Q000235:genetics / Q000378:metabolism*; D019656:Loss of Heterozygosity; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000378:metabolism*; D009368:Neoplasm Transplantation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D047368:Tumor Burden
chemical_list: C490098:LTF protein, human; D011994:Recombinant Proteins; D007781:Lactoferrin