LTF基因在鼻咽癌中的表达及其与甲基化的关系
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摘要
鼻咽癌是一种好发于我国南方各省及东南亚一带的上皮源性恶性肿瘤。流行病学及实验研究表明,鼻咽癌的发生是多阶段、多途径、多因素共同参与的复杂过程,遗传易感性、Epstein-Barr病毒(EBV)感染及包括饮食在内的环境因素等对鼻咽癌的发生起重要作用。在这些因素的共同作用下,鼻咽上皮细胞的鼻咽癌相关瘤基因和抑瘤基因发生遗传学变异和表遗传学改变的不断积累,最终引起细胞的恶性转化。
乳铁蛋白(lactotransferrin,LTF)是一种分泌性糖蛋白,可表达于多种组织中。一系列体内体外实验证明LTF具有多种生物学功能,它参与铁平衡、免疫调节、抗炎、抗肿瘤、止痛、调节骨代谢,参与胚胎发育和生殖等过程。本实验室前期工作通过分析鼻咽癌基因表达谱的结果选取了位于鼻咽癌高频缺失区3p21.3-22的LTF基因作为研究对象,探讨该基因与鼻咽癌发生发展的关系。实验结果显示,LTF基因在鼻咽癌组织(76%)中和7株鼻咽癌细胞系(100%)表达缺失或下调:原因分析显示其中63.6%、25%和30%的鼻咽癌组织及14%、14%和100%的鼻咽癌细胞系中分别存在启动子区DNA甲基化、微卫星位点LOH和基因突变。野生型LTF基因导入鼻咽癌细胞系5-8F后,稳定表达LTF基因的5-8F细胞细胞周期阻滞于G0-G1期,同时体外增殖能力和克隆形成能力下降。这些研究结果表明LTF基因很可能是一个与鼻咽癌关系密切的候选抑瘤基因。
在此基础上,本课题进一步分析了LTF基因的两个转录本LTF和△LTF在鼻咽癌组织中的表达。同时构建LTF基因P1和P2启动子甲基化与未甲基化的报告载体,并导入CNE2,观察启动子甲基化对LTF基因表达的调控作用。此外,野生型LTF基因被导入到鼻咽癌细胞系CNE2中并观察稳定表达LTF基因的CNE2细胞体内外生物学特性的影响,明确LTF基因在CNE2细胞中的作用。这些为探讨研究甲基化修饰与鼻咽癌组织LTF基因表达下调或缺失的相关性及LTF基因与鼻咽癌发生发展关系提供了进一步的证据。实验方法和结果如下:
首先,采用RT-PCR方法检测LTF基因两个转录本LTF和△LTF在34例原发性鼻咽癌和17例慢性鼻咽炎的组织表达,结果显示LTF和△LTF基因在慢性鼻咽炎组织中稳定表达,在82.35%(26/34)原发性鼻咽癌中表达缺失或下调。接着实验构建了LTF基因启动子甲基化和未甲基化的报告载体。启动子报告载体瞬时转染入6-10B和CNE2细胞,报告基因检测表明CNE2细胞比6-10B细胞具有相对较高的LTF启动子活性。定位于-99～+16区域启动子报告载体pGL3B/P1-116在6-10B和CNE2细胞中其报告基因都具有相对较高的活性,推测-99～+16区域为核心的启动子区。我们选择CNE2细胞做启动子regional methylation实验。SssI处理和对照的启动子连入报告载体并瞬时转染CNE2细胞,报告基因活性分析提示,与对照组相比,甲基化的LTF基因启动子活性明显降低,推测甲基化修饰抑制LTF启动子功能可能是NPC中LTF表达下调或缺失的重要原因。
此外,实验采用Origene公司含LTF基因cDNA序列的克隆载体pCMV6-XL5-LTF,并利用Not I酶切位点定向克隆到pcDNA3.1(-)载体中,构建了LTF基因的真核表达载体(pcsLTF)。利用脂质体将pcsLTF导入CNE2细胞,G418筛选获得抗性克隆,进一步采用RT-PCR检测LTF基因的mRNA表达,采用Western blotting检测LTF蛋白的表达,结果表明成功建立稳定表达LTF基因的CNE2细胞系(CNE2-LTF)。实验进一步检测了CNE2-LTF细胞的生物学特征:流式细胞分析结果表明,与对照组相比转染pcsLTF后可以使CNE2细胞阻滞于G1期,G0-G1期细胞比例明显增加(64.80%vs 55.13%),G2-M期(7.76%vs 15.13%)细胞比例减少。MTT的结果表明CNE2-LTF细胞的增殖速度明显低于对照组(P＜0.05);平板克隆形成实验结果显示CNE2-LTF细胞的克隆形成率明显低于对照组(37.5%vs 52.2%)。提示LTF基因稳定表达后导致CNE2细胞的增殖能力和克隆形成能力降低。裸鼠成瘤实验表明CNE2-LTF细胞肿瘤形成能力明显降低(P＜0.05)
本课题的实验结果表明,LTF基因两个转录本LTF与△LTF在原发性鼻咽癌表达缺失或下调;启动子区甲基化对LTF基因在鼻咽癌中表达下调或缺失可能起着重要作用;LTF基因稳定表达于CNE2细胞可明显改善其恶性生物学特性,使细胞阻滞于G1期,增殖能力和克隆形成能力降低,且裸鼠成瘤能力降低。综上所述,我们推测LTF基因很可能是鼻咽癌的候选抑瘤基因之一,LTF在鼻咽癌的发生发展中发挥重要作用,其具体作用还需要更多的体内外实验进行证实。
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.
乳铁蛋白(lactotransferrin,LTF)是一种分泌性糖蛋白,可表达于多种组织中。一系列体内体外实验证明LTF具有多种生物学功能,它参与铁平衡、免疫调节、抗炎、抗肿瘤、止痛、调节骨代谢,参与胚胎发育和生殖等过程。本实验室前期工作通过分析鼻咽癌基因表达谱的结果选取了位于鼻咽癌高频缺失区3p21.3-22的LTF基因作为研究对象,探讨该基因与鼻咽癌发生发展的关系。实验结果显示,LTF基因在鼻咽癌组织(76%)中和7株鼻咽癌细胞系(100%)表达缺失或下调:原因分析显示其中63.6%、25%和30%的鼻咽癌组织及14%、14%和100%的鼻咽癌细胞系中分别存在启动子区DNA甲基化、微卫星位点LOH和基因突变。野生型LTF基因导入鼻咽癌细胞系5-8F后,稳定表达LTF基因的5-8F细胞细胞周期阻滞于G0-G1期,同时体外增殖能力和克隆形成能力下降。这些研究结果表明LTF基因很可能是一个与鼻咽癌关系密切的候选抑瘤基因。
在此基础上,本课题进一步分析了LTF基因的两个转录本LTF和△LTF在鼻咽癌组织中的表达。同时构建LTF基因P1和P2启动子甲基化与未甲基化的报告载体,并导入CNE2,观察启动子甲基化对LTF基因表达的调控作用。此外,野生型LTF基因被导入到鼻咽癌细胞系CNE2中并观察稳定表达LTF基因的CNE2细胞体内外生物学特性的影响,明确LTF基因在CNE2细胞中的作用。这些为探讨研究甲基化修饰与鼻咽癌组织LTF基因表达下调或缺失的相关性及LTF基因与鼻咽癌发生发展关系提供了进一步的证据。实验方法和结果如下:
首先,采用RT-PCR方法检测LTF基因两个转录本LTF和△LTF在34例原发性鼻咽癌和17例慢性鼻咽炎的组织表达,结果显示LTF和△LTF基因在慢性鼻咽炎组织中稳定表达,在82.35%(26/34)原发性鼻咽癌中表达缺失或下调。接着实验构建了LTF基因启动子甲基化和未甲基化的报告载体。启动子报告载体瞬时转染入6-10B和CNE2细胞,报告基因检测表明CNE2细胞比6-10B细胞具有相对较高的LTF启动子活性。定位于-99～+16区域启动子报告载体pGL3B/P1-116在6-10B和CNE2细胞中其报告基因都具有相对较高的活性,推测-99～+16区域为核心的启动子区。我们选择CNE2细胞做启动子regional methylation实验。SssI处理和对照的启动子连入报告载体并瞬时转染CNE2细胞,报告基因活性分析提示,与对照组相比,甲基化的LTF基因启动子活性明显降低,推测甲基化修饰抑制LTF启动子功能可能是NPC中LTF表达下调或缺失的重要原因。
此外,实验采用Origene公司含LTF基因cDNA序列的克隆载体pCMV6-XL5-LTF,并利用Not I酶切位点定向克隆到pcDNA3.1(-)载体中,构建了LTF基因的真核表达载体(pcsLTF)。利用脂质体将pcsLTF导入CNE2细胞,G418筛选获得抗性克隆,进一步采用RT-PCR检测LTF基因的mRNA表达,采用Western blotting检测LTF蛋白的表达,结果表明成功建立稳定表达LTF基因的CNE2细胞系(CNE2-LTF)。实验进一步检测了CNE2-LTF细胞的生物学特征:流式细胞分析结果表明,与对照组相比转染pcsLTF后可以使CNE2细胞阻滞于G1期,G0-G1期细胞比例明显增加(64.80%vs 55.13%),G2-M期(7.76%vs 15.13%)细胞比例减少。MTT的结果表明CNE2-LTF细胞的增殖速度明显低于对照组(P＜0.05);平板克隆形成实验结果显示CNE2-LTF细胞的克隆形成率明显低于对照组(37.5%vs 52.2%)。提示LTF基因稳定表达后导致CNE2细胞的增殖能力和克隆形成能力降低。裸鼠成瘤实验表明CNE2-LTF细胞肿瘤形成能力明显降低(P＜0.05)
本课题的实验结果表明,LTF基因两个转录本LTF与△LTF在原发性鼻咽癌表达缺失或下调;启动子区甲基化对LTF基因在鼻咽癌中表达下调或缺失可能起着重要作用;LTF基因稳定表达于CNE2细胞可明显改善其恶性生物学特性,使细胞阻滞于G1期,增殖能力和克隆形成能力降低,且裸鼠成瘤能力降低。综上所述,我们推测LTF基因很可能是鼻咽癌的候选抑瘤基因之一,LTF在鼻咽癌的发生发展中发挥重要作用,其具体作用还需要更多的体内外实验进行证实。
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.
引文
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[24]Stokes K,Alston-Mills B,Teng C.Estrogen response element and the promoter context of the human and mouse lactoferrin genes influence estrogen receptor alpha-mediated transactivation activity in mammary gland cells.J Mol Endocrinol.2004,33(2):315-334.
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