LTF Expression In Nasopharyngeal Carcinoma And Its Relationship With Methylation

Nasopharyngeal carcinoma(NPC)is an epithelium-derived malignancy with high incidence in Southeast Asia and Southern China. The epidemiological and etiological studies indicate that the tumorigenesis of NPC is a multistage and multichannel process involving multiple factors.The various factors including genetic alterations, Epstein-Barr virus(EBV)infection,dietary and environmental factors are considered to contribute to the occurrence and development of NPC.On the backgroud of predisposed genetic and environmental factors, cumulative genetic and epigenetic alteration of NPC associated oneogenes and tumor suppressor genes(TSG)lead to malignant transformation of normal nasopharyngeal epithelial cells.Lactotransferrin(LTF)is a secretory glycoprotein expressed in a wide variety of tissues.The biological functions of LTF,which are confirmed in numerous in vitro and in vivo models,include participation in iron homeostasis,immunoregulatory properties,anti-inflammatory, anti-tumor,and analgesic actions,regulation of bone metabolism, participation in embryonic development,reproductive functions,and others.Based on the microarray experiments conducted on NPC and other human malignancies,LTF gene located at 3p21.3-22(which is a common deleted region in NPC)evoked our interest.Previous results in our laboratory have indicated that LTF was down-regulated or absent in 76%of NPC tissues and 100%of 7 NPC cell lines.To assess the possible molecular mechanism causing LTF inactivation in NPC cells,LOH,gene mutation and/or promoter methylation were detected in 63.6%,25%and 30%of NPC tissues an in 14%,14%and 100%of NPC cell lines respectively.Expression of LTF in 5-8F cells via gene transfeetion induce cell cycle arrest in G1 phase,much more slower proliferation and cloning efficiency.These results have shown that LTF may be a promising NPC-associated candidate TSG.Based on the previous work,we further analyzed the expression of two transcript isoforms of LTF in NPC tissues.Subsequently,We successfully constructed a series of methylated and unmethylated LTF gene P1 and P2 promoter recombinants containing luciferase reporter gene and analyzed the role of promoter methylation in regulating LTF gene expression in CNE2 cell line.In addition,transfection of LTF eukaryotic expression vector into CNE2 cells was performed by liposome method and the biological characteristic changes of transfected CNE2 cells were examined in vitro and in vivo for studying the possible roles of LTF in NPC.The experimental methods and results are as follows:To analyze the expression of two LTF isoforms at the transcription level,RT-PCR was performed.The expression of LTF in 35 primary NPC tissues and 17 chronic nasopharyngitis tissues were detected and the results showed that LTF was stably expressed in all the chronic nasopharyngitis tissues,but aberrantly expressed(absent or down-regulated)in 82.35%(26 of 34)of NPC tissues.Different P1 and P2 promoters were respectively cloned into pGL3-Basic vector without promoter and enhancer,which were confirmed by sequencing.P1 and P2 promoter recombinants were transiently transfected into 6-10B and CNE2 cell lines,lueiferase activities were measured.The results showed that LTF gene promoter exerted stronger activity in CNE2 cells than that in 6-10B.pGL3B/P1-116 recombinant mapping -99-+16 of LTF promoter exhibited relatively higher lueiferase activity in both 6-10B and CNE2 cells,suggesting -99-+16 region may be the core sequence of the P1 promoter.CNE2 cells were selected for regional methylation analysis of LTF promoters with reporter constructs.Promoter sequences methylated with SssI and controls prepared by similar treatment except the addition of SssI,were ligated back into the lueiferase reporter constructs and transfected into CNE2 cells,with pRL-SV40 cotransfection as an inner control.Our results indicated luciferase activities driven by methylated promoters were strikingly decreased compared to the control.It is inferred that suppression of LTF promoter function by methylation is an important reason leading to aberrant LTF expression in NPC.On the basis of pcDNA3.1(-)vector and pCMV6-XL5-LTF vector containing LTF cDNA sequence bought from Origene company,the eukaryotic LTF expression vector was consructed(designated pcs-LTF) and transfected into CNE2 cells by liposome method.After G418 selection,several G418-resistant cell clones were obtained.The stable expression of LTF both at mRNA and protein levels in the G418-resistant cell clones was detected by RT-PCR and Western blotting respectively. The results showed that the CNE2 cell line stably expressing LTF gene (named CNE2-LTF)has been successfully established.In vitro and in vivo experiments illustrated that biological characteristics of CNE2-LTF cells changed significantly.Flow cytometry (FCM)analysis showed that LTF expression could block the cell cycle progression of CNE2-LTF cells in G1 phase,leading to an increase in the number of cells in G1 phase(64.80%vs 55.13%)and a decrease in the number of cells in G2/M phase(7.76%vs 15.13%).MTT analysis and colony formation assay showed that,compared with CNE2 cells transfeeted with blank vector(named CNE2-pc3.1),CNE2-LTF cells proliferated much more slowly(P＜0.05)and had a much lower cloning efficiency(37.5%vs 52.2%),providing evidence that LTF had an inhibitory effect on proliferation of CNE2-LTF cells.In order to compare the ability of tumor formation between CNE2-LTF cells and CNE2-pc3.1, we inoculated subeutaneouly 5×10~6 cells of each kind to six nude mice respectively.Tumor formation was examined after 6 weeks.All the nude mice were alive with the tumor developed.CNE2-LTF cells manifested much weaker tumor formation potential compared to CNE2-pc3.1 cells (P＜0.05).Above all,our results demonsrate that expression of LTF two isoforms is absent or down-regulated in NPC;methylation of LTF promoter plays an important role in inactivation of LTF gene;sequences mapping -99- +16 of LTF gene may be the core site of the promoter; stable expression,of LTF could induce cell cycle arrest in G1 phase, decrease the proliferation and cloning abilites of CNE2-LTF cells,and induce tumor suppression in nude mice in vivo.All these data confirm that LTF can be a candidate TSG in NPC.The detailed mechanism of LTF in the tumorigenesis of NPC remains to be further studied.