染色体3p21鼻咽癌易感基因区LTF基因的筛查及其抑制鼻咽癌细胞增殖的功能研究
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摘要
【染色体3p21鼻咽癌易感基因区LTF基因的筛查】
在前期研究中，我室利用18个湖南鼻咽癌高发家系，通过连锁分析，首次发现染色体3p21部分区域与湖南家族性鼻咽癌发病紧密连锁，经单倍型分析和精细定位，将鼻咽癌易感基因区域定位于微卫星标记D3S1289-D3S1298之间。此结果提示：染色体3p21区域可能存在鼻咽癌的候选易感基因。
为了进一步筛查与鉴定染色体3p21区域鼻咽癌的候选易感基因，本研究制备了两种不同的基因芯片，一种是基于鼻咽癌抑制消减杂交文库的cDNA mciroarray，另一种是包含染色体3p21区域(D3S1289-D3S1298)288个基因的专制芯片。为提高芯片筛查结果的可靠性，我们运用两种不同的基因芯片对相同的试验样本进行研究。
首先，以4个鼻咽癌相关的抑制消减杂交文库(NNTDH12、TRP19 DHNE1、TNPC DNN119和TNN119 DNPC)为模板，利用PCR技术扩增点制cDNA芯片所需的克隆，每个文库约300个、总共1200余个克隆。克隆纯化后点制基于鼻咽癌抑制消减杂交文库的cDNA mciroarray。
10例鼻咽慢性炎症组织和19例鼻咽癌组织标本以及3例鼻咽癌细胞样本与定制好的cDNA芯片杂交，基本数据利用Genepix Pro 3.0(Axon Instruments)来收集和基础处理。数据分析前，去掉芯片中的污点／坏点。为降低由于低表达基因点所造成的实验偏倚，只选择Cy5和Cy3信号值皆大于200的基因点，并对符合条件基因点的信号值进行均一化处理。经过上述处理后，共获得1097个有效克隆信号值。
对上述已预处理的芯片杂交数据，挑取在所有样本中存在＞85％的信号值的克隆作进一步分析，898个(898／1097)克隆通过滤过。使用Euclidean distance and average linkage clustering方法，对每个样本和每个基因取平均值和均化处理，然后Hierarchical clustering聚类。聚类结果显示：898个克隆能够把鼻咽慢性炎症组织和鼻咽肿瘤组织很清晰的分为两大类，T01_F，T01_S作为两重复样本聚在一起，N8，N9和N10样本因均为鼻咽慢性炎症上皮，聚为一小束，再与其他正常鼻咽上皮聚类；三种鼻咽癌细胞系C1，C2和C3聚在一起，再与其他鼻咽癌组织样本聚为一大类。
虽然898个克隆能够较好的区分鼻咽慢性炎症组织和鼻咽肿瘤组织，但是我们希望能够用最少的克隆数来达到最大的检验效能(即区分鼻咽慢性炎症组织和鼻咽肿瘤组织)。于是利用Genesis软件的ANOVA功能(选择p＜0.001为统计差异标准)对898个克隆进行了筛选，其中105个克隆满足要求。利用105个差异表达克隆进行Hierarchical clustering聚类。聚类结果表明：105个克隆仍然能够很好地把鼻咽慢性炎症组织和鼻咽肿瘤组织很清晰的分为两大类，T01_F，T01_S作为两重复样本聚在一起，N8，N9和N10样本因均为鼻咽慢性炎症上皮，先聚为一小类，再与其他正常鼻咽上皮聚类；三种鼻咽癌细胞系C1，C2和C3聚在一起，再与其他鼻咽癌组织样本进行聚类，说明该105个克隆能够达到898个克隆相同的检验效能。
为进一步鉴定上述105个差异表达克隆，利用PCR方法扩增出相应的克隆片段，然后将PCR产物纯化后送生物技术公司测序，测序结果与NCBI数据库进行同源性比对(http://www.ncbi.nlm.nih.gov/BLAST)。通过同源比对发现37个在鼻咽癌组织中高表达的克隆实际上代表STAT5A，RAB25、SPARC以及SPRR3等14个已知基因。各基因出现的频率存在差异，以STAT5A基因出现的频率最高(为7次，占18.92％)，其次是RAB25基因(出现5次，占13.51％)，紧追其后为SPARC和SPRR3基因。这14个基因分布在9条染色体上(包括1q、2q、4p、5q、7p、7q、12p、12q、13q、15q以及17q)。另外68个在鼻咽癌组织中低表达的克隆实际上代表LTF，PLUNC、CDC37L1以及HNRPDL等18个已知基因。各基因出现的频率存在差别，以PLUNC基因出现的频率最高(为21次，占30.88％)，其次是CDC37L1基因(出现9次，占13.24％)，紧追其后为HNRPDL基因。这18个基因分布在12条染色体上(包括1q、3p、4q、7q、8q、9p、11q、12p、12q、14q、16p、20q以及22q)。至此，利用基于鼻咽癌抑制消减杂交文库的cDNA microarray筛查到了包括染色体3p21鼻咽癌易感基因区LTF基因在内的32个鼻咽癌差异表达基因。
此外，我们利用包含染色体3p21区域288个基因的专制芯片对上述相同的实验样本进行了研究，发现在3p21区域288个基因中，4个基因在鼻咽癌中表达下调，为乳铁传递蛋白LTF，dystroglycan 1，FLJ34969，和TU3A基因，而glutamate receptor，metabotropic 2，interleukin 17receptor B在鼻咽癌中存在表达上调。
综合分析两组结果，发现LTF基因在两种基因芯片中得到相同的表达变化——在鼻咽癌组织中表达下调。两种不同的基因芯片、相同的研究样本，得到了相同的表达变化，有理由相信LTF基因在鼻咽癌发生、发展中起着重要的作用。
为了验证芯片结果的可靠性，利用real-time quantitative RT-PCR技术，选择LTF、PLUNC、CDC37L1以及HNRPDL等4个在鼻咽癌组织中低表达的基因，另外选择在鼻咽癌组织中高表达的STAT5A、RAB25和SPARC基因进行验证。结果显示：LTF、PLUNC、CDC37L1和HNRPDL基因在鼻咽癌组织表达下调，STAT5A、RAB25和SPARC基因在鼻咽癌中表达上调。与芯片结果一致，说明芯片结果是可靠的。
为了在大样本中进一步验证基因芯片筛查出来的差异表达基因，本研究利用组织芯片技术(TMA)与原位杂交技术结合验证LTF，PLUNC和STAT5A基因的表达情况。结果发现：LTF和PLUNC基因在鼻咽癌中的阳性表达率均明显低于癌旁上皮和鼻咽慢性炎症上皮(p＜0.01)，而STAT5A基因在鼻咽癌中的阳性表达率明显高于癌旁上皮和鼻咽慢性炎症上皮(p＜0.01)。此结果与芯片结果一致，进一步说明了芯片结果的可靠性。
【LTF基因抑制鼻咽癌细胞增殖的功能研究】
LTF(Lactotransferrin)基因能够在两种基因芯片中得到相同的表达变化，结合本室前期的家系连锁分析结果，有理由相信位于染色体3p21鼻咽癌易感基因区的LTF基因在鼻咽癌发病中起着重要作用。同时曾朝阳博士运用组织微阵列(TMA)和免疫组织化学技术对LTF基因蛋白水平的表达情况在较大样本中进行了研究，发现Lactotransferrin在鼻咽癌中表达下调，鼻咽癌癌旁和鼻咽慢性炎症组织中存在高表达。几乎所有的鼻咽部腺体均能检测到Lactotransferrin蛋白的存在，证实Lactotransferrin蛋白在腺体中高表达、为一细胞分泌性蛋白。此外，Lactotransferrin蛋白的表达与鼻咽癌转移呈负相关(p＜0.05)。Lactotransferrin蛋白的表达与鼻咽癌临床分期存在显著相关性(p＜0.01)。临床Ⅰ期和Ⅱ期鼻咽癌中Lactotransferrin蛋白的表达明显高于临床Ⅲ和Ⅳ期(p＜0.01)。提示它可能与鼻咽癌的侵袭转移和临床进展有关，为鼻咽癌候选易感基因，可作为鼻咽癌侵袭、转移和临床进展预测的分子靶标。
基因芯片和组织芯片结果均发现LTF基因在鼻咽癌中表达明显下调。为了明确LTF基因抑制鼻咽癌发生发展的可能作用机制，下一步拟探讨LTF基因对体外鼻咽癌细胞株的影响。
首先利用重组人乳铁传递蛋白(Recombinant human lactoferrin，rhlf)对鼻咽癌细胞株CNE1、5-8F、HNE1以及6-10B进行干预，MTT试验检测LTF基因对体外鼻咽癌细胞株生长的影响。结果发现LTF基因能够明显抑制鼻咽癌细胞CNE1、5-8F以及HNE1的生长。CNE1、5-8F以及HNE1细胞用10μmol／L rhlf干预后生长速度明显减慢(与未干预组比较)，干预24或48小时后即出现统计学差异。此结果再次从体外细胞角度验证了本论文第一部分的基因芯片和组织芯片结果。表明LTF不仅在鼻咽癌组织中表达明显下调，而且能够延缓体外鼻咽癌细胞株生长、抑制肿瘤形成。
然后，利用10μmol／L的rhlf对鼻咽癌细胞株ENE1、5-8F、HNE1以及6-10B进行干预，通过流式细胞术检测分析LTF基因对体外鼻咽癌细胞株细胞周期的影响。结果发现CNE1、5-8F以及HNEl细胞经10μmol／L的rhlf干预24或48小时后GO-G1期细胞百分比明显增多；S期和G2-M期细胞百分比则减少。但10μmol／L的rhlf干预对鼻咽癌6-10B细胞未产生明显影响。6-10B细胞是四种鼻咽癌细胞株中唯一一株没有转移潜能的，rhlf干预对其生长未产生明显的影响，这与前述基于组织微阵列(TMA)的免疫组织化学结果(LTF基因可能与鼻咽癌的侵袭转移和临床进展有关)互相印证，结果再次提示LTF主要作用于鼻咽癌发生发展的中晚期、在鼻咽癌转移过程中应该具有重要作用。此结果与MTT试验结果基本一致，证实LTF基因能够明显增加G0-G1期细胞比例、抑制体外鼻咽癌细胞的生长、增殖。
为了探讨LTF基因延缓G0-G1期进程、抑制鼻咽癌细胞生长的可能机制，采用具有高转移特性的鼻咽癌5-8F细胞作为研究对象，应用rhlf对其进行干预，利用western blot技术检测Cyclin D1，p-Rb，p21，p27，p53，总stat3，p-star3(Tyr705)以及MAPK信号传导通路中JNK2，c-Jun，总ERK1／2，p-ERK1／2，c-fos等相关信号分子干预前后的表达情况，分析LTF基因抑制体外鼻咽癌细胞增殖的作用机制。
鼻咽癌5-8F细胞用10μmol／L rhlf分别干预0min，5 min，10 min，30 min，60 min，12h，24h，36h，48h和72h后，抽提蛋白，western blot检测细胞周期蛋白cyclin D1、p-Rb的表达情况，结果发现：5-8F细胞用10μmol／L rhlf干预后周期蛋白cycl in D1、p-Rb的表达均低于对照组，说明LTF基因对周期蛋白cyclin D1、p-Rb的表达有重要影响。在此基础上，又检测了两种细胞周期素激酶(CDK)抑制蛋白p21，p27的表达情况。结果显示：鼻咽癌5-8F细胞用10μmol／L rhlf分别干预5 min，10 min，30 min，60 min，12h，24h，36h，48h和72h后，细胞周期素激酶(CDK)抑制蛋白p21，p27的表达与对照组相比(即0min)均增加。上述结果提示LTF基因通过调节周期蛋白cyclin D1、p-Rb以及细胞周期素激酶的表达来影响鼻咽癌5-8F细胞的增殖。
为了进一步了解引起cyclin D1、p-Rb改变的相关分子事件，根据已有试验结果、结合文献资料，我们选择MAPK信号传导通路的信号分子用western blot方法对其表达情况进行检测。鼻咽癌5-8F细胞用10μmol／Lrhlf分别干预5 min，10 min，30 min，60 min，12h，24h，36h，48h和72h后，抽提蛋白，western blot检测JNK2，c-Jun，总ERK1／2，p-ERK1／2和c-fos的表达情况。结果显示：LTF对总ERK1／2的表达未见明显影响；但降低p-ERK1／2的表达，用10μmol／L rhlf干预60min后其表达量明显减少，随着干预时间的延长，其表达量有进一步减少的趋势：LTF对JNK2蛋白表达产生影响，用10μmol／L rhlf干预12h后JNK2的表达量明显减少；同时LTF降低c-Jun的表达，用10μmol／L rhlf干预30min后即出现明显减少，随后就维持在一个较为平稳的水平；此外LTF对c-fos的表达有着较大影响，用10μmol／L rhlf干预60min后其表达量明显减少，随着干预时间的延长，其表达量有进一步减少的趋势，这种变化与p-ERR1／2的表达变动情况相吻合。
综上所述，LTF基因通过负性调控MAPK信号传导通路的JNK2和ERR亚家族，下调细胞周期蛋白cyclin D1，p-Rb以及细胞周期素激酶的表达，延缓鼻咽癌细胞G0-G1期进程，最终通过调节cyclins／CDKs／CKI／pRb为核心的G1／S期细胞周期网络调控系统来调控鼻咽癌5-8F细胞的生长、增殖。当然，基因表达调控系统是非常复杂和有机联系的，不同分子以及不同通路之间存在cross—talk，要明确LTF基因在其中所起的真正作用，还需以后进一步的实验证实。
【Identification of LTF Gene on the Region of NPC Susceptibility Gene—Chromosome 3p21】
In our previous study, linkage analysis of 18 pedigrees from Hunan Province of China suggests that potential susceptibility loci linked to NPC are located on chromosome 3p21. The putative NPC susceptibility locus is located a region from D3S1289 to D3S1298 on chromosome 3p21 through haplotype analysis. These results show that the candidate NPC susceptibility genes may be on chromosome 3p21.
To scan and identify the candidate NPC susceptibility genes on chromosome 3p21 further, two different gene chips were manufactured. One was cDNA microarray of subtracted cDNA libraries constructed by suppression subtractive hybridization, the other was a custom-made chromosome 3p21-specific cDNA microarray. Two different cDNA microarray were used to scran the same samples for improving their reliability.
First, the colonies used to made cDNA microarray were prepared by PCR from 4 NPC's subtracted cDNA libraries (NNTDH12, TRP19 DHNE1, TNPC DNN119, and TNN119 DNPC). There were more than 1,200 colonies including 300 colonies from every library to produce cDNA microarray. The purified colonies were used to made cDNA microarray of NPC's subtracted cDNA libraries.
After 19 NPCs, 3 NPC derived cell lines, and 10 chronic inflammation of nasopharyngeal mucosa tissue samples hybridized the cDNA microarray, primary data collection and analysis were carrded out via Genepix Pro 3.0 (Axon Instruments). The area of the array with obvious blemish was manually flagged and excluded from subsequent analysis. To minimize artifacts arising from low expression values, only genes with raw intensity values for both Cy3 and Cy5 of＞200 counts were chosen for differential analysis. Then overall intensifies were normalized with a correction coefficient obtained using the ratios of these genes. One thousand and ninety-seven valid colonies were obtained.
For the above preprocessed hierarchical clustering, colonies for which＞85% of measurements among all the samples were selected for further analysis. Altogether 898 of the 1,097 colonies passed this variation filter for the initial, then mean-centered and normalized with respect to other colonies in the sample and corresponding colonies in other samples. First, this list of colonies was used in Genesis software (http://genome.tugraz.at) to perform a hierarchical clustering analysis using the Euclidean distance and average linkage clustering. The specimens were divided into two subtypes based on these colonies: the NPC groups versus the chronic inflammation of nasopharyngeal mucosa groups. Within the "NPC" branch of the dendrogram, 3 NPC_derived cell lines were clustered as a small branch of NPC. T01_S was the replicate sample of the T01_F sample, and they were clustered tightly.
Though chronic inflammation of nasopharyngeal mucosa tissue and nasopharyngeal carcinoma tissue samples could be distinguished by 898 colonies, we hoped to find the smallest colony number to reach the aim. Genesis software was used for the following in ANOVA analysis to obtain the differentially expressed colonies among chronic inflammation of nasopharyngeal mueosa, NPCs and NPC-dedved cell lines. We obtained 105 differentially expression colonies. At last, these differentially expressed colonies were used in Genesis software to perform a hierarchical clustering analysis using the Euclidean distance and average linkage clustering. The specimens were divided into two subtypes based on these colonies: the NPC groups versus the chronic inflammation of nasopharyngeal mueosa groups. Within the "NPC" branch of the dendrogram, 3 NPC_derived cell lines were clustered as a small branch of NPC. T01_S was the replicate sample of the T01_F sample, which was clustered tightly. It showed that 105 colonies had the same test efficiency.
To identify the 105 colonies differentially expressed in NPC, all the cDNA colonies were amplified by using of the SSH secondary primers. The PCR products were purified from the gel anddirectly sequenced. The cDNA sequences were compared to GenBank by Blast search (http://www.nebi.nlm.nih.gov/BLAST/). There were 14 known genes, including STAT5A, RAB25, SPARC, and SPRR3, et al. to be identified in all the 37 colonies expressed highly in NPC and NPC_derived cell lines. Different frequency exists in every gene. STAT5A gene had the highest frequency of 18.92%, the second was RAB25(13.51%), followed by SPARC, SPRR3 and so on. These 14 genes were distributed on 9 chromosomes, including 1q, 2q, 4p, 5q, 7p, 7q, 12p, 12q, 13q, 15q and 17q. Eighteen known genes, including LTF, PLUNC, CDC37L1, HNRPDL genes, et al. were identified in 68 colonies which expressed poorly in N-PC and NPC_derived cell lines. The frequency to every gene was different. PLUNC gene had the highest frequency of 30.88%, the second was CDC37L1 (13.24%), followed by HNRPDL and so on. These 18 genes were distributed on 12 chromosomes, including lq, 3p, 4q, 7q, 8q, 9p, 11q, 12p, 12q, 14q, 16p, 20q and 22q. We scanned 32 genes differentially expressed in NPC using the cDNA mieroarray of subtracted cDNA libraries constructed by SSH, including LTF gene on the region of NPC susceptibility gene—ehromosome 3p21.
In addition, the results showed that lactotransferrin (LTF), dystroglyean 1, FLJ34969, and TU3A genes were down-regulated in NPC samples, glutamate receptor, metabotropie 2(GRM2) and interleukin 17 receptor B (IL17RB) were up-regulated in NPC samples by a custom-made chromosome 3p21-specifie cDNA mieroarray including 288 genes in this region in the same samples.
Through eompositive analysis of these results, we found that LTF was down-regulated in NPC samples in the two different gene chips. There was the parallel change in the same samples by different cDNA mieroarray. So we could believe that LTF gene might play an important role in pathogenesis of NPC.
To confirm the cDNA mieroarray results, we chose the same samples of cDNA mieroarray to perform real-time quantitative RT-PCR of LTF, PLUNC, CDC37L1, and HNRPDL genes, which were down-regulated in NPC samples, and the same was done to STAT5A, RAB25, and SPARC genes, which were up-regulated in NPC samples. The expression of LTF, PLUNC, CDC37L1, and HNRPDL genes were low in NPC and the expression of STAT5A, RAB25 and SPARC genes were high in NPC. It corresponded with the results of cDNA mieroarray and proved its reliability.
To validate the genes differentially expressed in NPC by cDNA mieroarray, we used in situ hybridization technique to confirm once more the expression of PLUNC, CDC37L1 and STAT5A genes with two NPC-tissue arrays. The positive expression rate of LTF and PLUNC genes in the NPC adjacent epitheliums and chromo nasopharyngeal was significantly higher than that of NPCs (p＜0.01). The positive expression rate of STAT5A gene in the NPC adjacent epitheliums and chromo nasopharyngeal was significantly lower than that of NPCs (p＜0.01). It corresponded with the results of cDNA microaray and proved its reliability.
【Function Study of LTF Gene that Inhibited Proliferation in the Nasopharyngeal Carcinoma Cells】
LTF was the parallel change in the different cDNA mieroarray. With compositive analysis of it and the previous results by linkage analysis of pedigrees, we had reasons to believe that LTF gene might play an important role in pathogenesis of NPC. At the same time, the expression of lactotransferrin protein was studied using immunoehemisty in NPC-tissue microarray by Dr. Zhao-yang ZENG. The results showed that laetotransferrin protein was down-regulated in NPC and up-regulated in NPC adjacent epitheliums and chronic inflammation of nasopharyngeal mueosas. Laetotransferrin protein could be detected in almost all of gland in nasopharynx, confirming that lactotransferrin protein was a secretory protein up-regulated in nasopharynx. In addition, the expression of laetotransferfin protein was negative correlated to the neck metastasis states (p＜0.05) and was significantly correlated to the clinical stages of NPC (p＜0.01). The expression of lactotransferrin protein in clinical stageⅠandⅡis obviously higher than that of the clinical stageⅢandⅣ. These findings suggest that LIF may be correlated with invading, metastasis and clinical evlovement and it is a good candidate tumor suppressor gene in NPC, and it may be a molecular marker for prediction of NPC invading, metastasis and clinical evlovement.
The results of cDNA mieroarray and TMA showed that LTF gene was down-regulated in NPC samples. To investigate the mechanism of LTF to NPC, we investigated the effects of LTF in the Nasopharyngeal Carcinoma Cells in vitro.
First, we used recombinant human laetoferrin (rhlf) to treat the NPC cell lines: CNE1, 5-8F, HNE1, and 6-10B. Then, we chose the reduction of the yellow tetrazolium salt3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay to assessment of proliferation- suppressing effect of LIF in NPC cell lines. The results of MTT assay showed that LTF significantly inhibited 5-8F, CNE1, and HNE1 cell proliferation after treated with lactoferrin. The growth of 5-8F, CNE1, and HNE1 cell treated with laetoferrin was suppressive compared with the control group. It was significantly different after treated with laetoferrin for 24 hours and 48 hours. It validated the results of cDNA microarray and TMA in part I from NPC cell lines in vitro. These findings suggested that LTF could reduce the growth of NPC cell lines in vitro and suppress the development of tumor, in addition, LTF was down-regulated in NPC samples.
To follow, we chose 10μmol/L recombinant human lactoferrin (rhlf) to treat the NPC cell lines: CNE1, 5-8F, HNE1, and 6-10B. Then, the experiment of flow cytometry was applied for cell cycle analysis of NPC cell lines to evaluate the effect of LTF. The findings showed that the percentage of G0-G1 phase of the cell cycle in 5-8F, CNE1, and HNE1 cells was significantly increased after treated with lactoferrin (10μmol/L) for 24 hours or 48 hours, but the percent of G2-M phase and S phase was reduced respectively. But it had no significantly effect to 6-10B cell after treated with lactoferrin. Among 5-8F, CNE1, HNE1, and 6-10B cells, 6-10B cell was the only one that had no characteristic of metastasis. It corresponded with the results of immunochemisty in NPC-tissue microarray- LTF might be correlated with invading, metastasis and clinical evolvement. The results hinted again that LTF mainly functioned in the mid-terminal stages of NPC and played an important role in the stages. These data were consistent with results of MTT army. Moreover, it proved that LTF could suppress the growth and proliferation of NPC cells in vitro.
To reveal underlying mechanism that LTF delayed G0-G1 phase progress to inhibit growth of NPC cells in vitro, we chose 5-8F cell line with high characteristic of metastasis as investigation object. After 5-8F cells were treated with lactoferrin, we examined the expression levels of Cyclin D1, p-Rb, p21, p27, p53, total star3, p-stat3(Tyr705) and signal molecules of mitogen-activated protein kinase (MAPK) signal pathway including JNK2, c-Jun, total ERK1/2, p-ERK1/2, and c-fos by western-blot technique. At last, we hoped to reveal some underlying mechanism that LTF inhibited the growth and proliferation of NPC cells in vitro.
After NPC derived cell lines 5-8F was treated with lactoferrin (10μmol/L) for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, we prepared their protein in turn and tested the expression levels of cyclin D1 and phosphorylated Rb by western blot. After treated with lactoferrin, the expression levels of cyclin D1 and phosphorylated Rb were reduced distinctly compared with the corresponding levels in the untreated. The results suggested that LTF affected the expression of cyclin D1 and p-Rb. Subsequently, we studied the expression of p21 and p27 proteins that were cyclin-dependent kinase inhibitor. After treated with lactoferrin for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, the expression levels of p21 and p27 proteins were increased significantly compared with the corresponding levels in the untreated. In conclusion, these findings suggested that LTF could suppress the proliferation of NPC derived cell lines 5-8F through regulating the moeulars of cyclin D1, p-Rb, cyclin-dependent kinase, and cyclin-dependent kinase inhibitor.
To additionally realize the molecular changes associated with cyclin D1 and p-Rb, we examined the signal molecules of mitogen-activated protein kinase (MAPK) signal pathway by western blot according to our previous results and accumulated document evidences. After NPC derived cell lines 5-8F was treated with lactoferrin (10μmol/L) for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, we prepared their protein in turn and tested the expression levels of JNK2, c-Jun, total ERK1/2, p-ERK1/2, and c-fos by western-blot technique. The expression level of p-ERK1/2 was reduced significantly after treated with lactoferrin (10μmol/L) for 60min. There was a decreasing trend of expression quantity with extension of treatment time. But we found that LTF gene had no effect on the expression level of total ERK. The expression level of JNK2 was reduced obviously after treated with lactoferrin (10μmol/L) for 12h. LTF gene affected the expression level of JNK2 protein. LTF gene cut down the expression levels of not only JNK2 protein but also c-Jun protein. The expression level of c-Jun was decreased obviously after treated with lactoferrin (10μmol/L) for 30min, and then was kept at an invariablenes level. In addition, LTF gene had an important effect on the expression of c-fos. The expression level of c-fos was reduced significantly after treated with lactoferrin (10μmol/L) for 60min. There was a decreasing trend of expression quantity with extension of treatment time. It corresponded with the changes ofp-ERK1/2.
In summary, LTF negatively modulated sub-families of JNK2, and ERK of MAPK signal pathway to suppress the expression of cyelin D1, p-Rb, and Cyelin-Dependent Kinase and to delay the G0-G1 progression of nasopharyngeal carcinoma cells. LTF inhibited growth and proliferation of 5-8F cells in vitro by modulating the G1/S phase regulation systems with regulating eyelins/CDKs/CKI/pRb as the key point. Subsequently, regulation systems of gene expression are very complicated and organic contacted. There are some cross-talks among different pathways anddifferent moleeulars. To reveal the reality ftmetions of LTF, we must do more experiments to verify it farther.
在前期研究中，我室利用18个湖南鼻咽癌高发家系，通过连锁分析，首次发现染色体3p21部分区域与湖南家族性鼻咽癌发病紧密连锁，经单倍型分析和精细定位，将鼻咽癌易感基因区域定位于微卫星标记D3S1289-D3S1298之间。此结果提示：染色体3p21区域可能存在鼻咽癌的候选易感基因。
为了进一步筛查与鉴定染色体3p21区域鼻咽癌的候选易感基因，本研究制备了两种不同的基因芯片，一种是基于鼻咽癌抑制消减杂交文库的cDNA mciroarray，另一种是包含染色体3p21区域(D3S1289-D3S1298)288个基因的专制芯片。为提高芯片筛查结果的可靠性，我们运用两种不同的基因芯片对相同的试验样本进行研究。
首先，以4个鼻咽癌相关的抑制消减杂交文库(NNTDH12、TRP19 DHNE1、TNPC DNN119和TNN119 DNPC)为模板，利用PCR技术扩增点制cDNA芯片所需的克隆，每个文库约300个、总共1200余个克隆。克隆纯化后点制基于鼻咽癌抑制消减杂交文库的cDNA mciroarray。
10例鼻咽慢性炎症组织和19例鼻咽癌组织标本以及3例鼻咽癌细胞样本与定制好的cDNA芯片杂交，基本数据利用Genepix Pro 3.0(Axon Instruments)来收集和基础处理。数据分析前，去掉芯片中的污点／坏点。为降低由于低表达基因点所造成的实验偏倚，只选择Cy5和Cy3信号值皆大于200的基因点，并对符合条件基因点的信号值进行均一化处理。经过上述处理后，共获得1097个有效克隆信号值。
对上述已预处理的芯片杂交数据，挑取在所有样本中存在＞85％的信号值的克隆作进一步分析，898个(898／1097)克隆通过滤过。使用Euclidean distance and average linkage clustering方法，对每个样本和每个基因取平均值和均化处理，然后Hierarchical clustering聚类。聚类结果显示：898个克隆能够把鼻咽慢性炎症组织和鼻咽肿瘤组织很清晰的分为两大类，T01_F，T01_S作为两重复样本聚在一起，N8，N9和N10样本因均为鼻咽慢性炎症上皮，聚为一小束，再与其他正常鼻咽上皮聚类；三种鼻咽癌细胞系C1，C2和C3聚在一起，再与其他鼻咽癌组织样本聚为一大类。
虽然898个克隆能够较好的区分鼻咽慢性炎症组织和鼻咽肿瘤组织，但是我们希望能够用最少的克隆数来达到最大的检验效能(即区分鼻咽慢性炎症组织和鼻咽肿瘤组织)。于是利用Genesis软件的ANOVA功能(选择p＜0.001为统计差异标准)对898个克隆进行了筛选，其中105个克隆满足要求。利用105个差异表达克隆进行Hierarchical clustering聚类。聚类结果表明：105个克隆仍然能够很好地把鼻咽慢性炎症组织和鼻咽肿瘤组织很清晰的分为两大类，T01_F，T01_S作为两重复样本聚在一起，N8，N9和N10样本因均为鼻咽慢性炎症上皮，先聚为一小类，再与其他正常鼻咽上皮聚类；三种鼻咽癌细胞系C1，C2和C3聚在一起，再与其他鼻咽癌组织样本进行聚类，说明该105个克隆能够达到898个克隆相同的检验效能。
为进一步鉴定上述105个差异表达克隆，利用PCR方法扩增出相应的克隆片段，然后将PCR产物纯化后送生物技术公司测序，测序结果与NCBI数据库进行同源性比对(http://www.ncbi.nlm.nih.gov/BLAST)。通过同源比对发现37个在鼻咽癌组织中高表达的克隆实际上代表STAT5A，RAB25、SPARC以及SPRR3等14个已知基因。各基因出现的频率存在差异，以STAT5A基因出现的频率最高(为7次，占18.92％)，其次是RAB25基因(出现5次，占13.51％)，紧追其后为SPARC和SPRR3基因。这14个基因分布在9条染色体上(包括1q、2q、4p、5q、7p、7q、12p、12q、13q、15q以及17q)。另外68个在鼻咽癌组织中低表达的克隆实际上代表LTF，PLUNC、CDC37L1以及HNRPDL等18个已知基因。各基因出现的频率存在差别，以PLUNC基因出现的频率最高(为21次，占30.88％)，其次是CDC37L1基因(出现9次，占13.24％)，紧追其后为HNRPDL基因。这18个基因分布在12条染色体上(包括1q、3p、4q、7q、8q、9p、11q、12p、12q、14q、16p、20q以及22q)。至此，利用基于鼻咽癌抑制消减杂交文库的cDNA microarray筛查到了包括染色体3p21鼻咽癌易感基因区LTF基因在内的32个鼻咽癌差异表达基因。
此外，我们利用包含染色体3p21区域288个基因的专制芯片对上述相同的实验样本进行了研究，发现在3p21区域288个基因中，4个基因在鼻咽癌中表达下调，为乳铁传递蛋白LTF，dystroglycan 1，FLJ34969，和TU3A基因，而glutamate receptor，metabotropic 2，interleukin 17receptor B在鼻咽癌中存在表达上调。
综合分析两组结果，发现LTF基因在两种基因芯片中得到相同的表达变化——在鼻咽癌组织中表达下调。两种不同的基因芯片、相同的研究样本，得到了相同的表达变化，有理由相信LTF基因在鼻咽癌发生、发展中起着重要的作用。
为了验证芯片结果的可靠性，利用real-time quantitative RT-PCR技术，选择LTF、PLUNC、CDC37L1以及HNRPDL等4个在鼻咽癌组织中低表达的基因，另外选择在鼻咽癌组织中高表达的STAT5A、RAB25和SPARC基因进行验证。结果显示：LTF、PLUNC、CDC37L1和HNRPDL基因在鼻咽癌组织表达下调，STAT5A、RAB25和SPARC基因在鼻咽癌中表达上调。与芯片结果一致，说明芯片结果是可靠的。
为了在大样本中进一步验证基因芯片筛查出来的差异表达基因，本研究利用组织芯片技术(TMA)与原位杂交技术结合验证LTF，PLUNC和STAT5A基因的表达情况。结果发现：LTF和PLUNC基因在鼻咽癌中的阳性表达率均明显低于癌旁上皮和鼻咽慢性炎症上皮(p＜0.01)，而STAT5A基因在鼻咽癌中的阳性表达率明显高于癌旁上皮和鼻咽慢性炎症上皮(p＜0.01)。此结果与芯片结果一致，进一步说明了芯片结果的可靠性。
【LTF基因抑制鼻咽癌细胞增殖的功能研究】
LTF(Lactotransferrin)基因能够在两种基因芯片中得到相同的表达变化，结合本室前期的家系连锁分析结果，有理由相信位于染色体3p21鼻咽癌易感基因区的LTF基因在鼻咽癌发病中起着重要作用。同时曾朝阳博士运用组织微阵列(TMA)和免疫组织化学技术对LTF基因蛋白水平的表达情况在较大样本中进行了研究，发现Lactotransferrin在鼻咽癌中表达下调，鼻咽癌癌旁和鼻咽慢性炎症组织中存在高表达。几乎所有的鼻咽部腺体均能检测到Lactotransferrin蛋白的存在，证实Lactotransferrin蛋白在腺体中高表达、为一细胞分泌性蛋白。此外，Lactotransferrin蛋白的表达与鼻咽癌转移呈负相关(p＜0.05)。Lactotransferrin蛋白的表达与鼻咽癌临床分期存在显著相关性(p＜0.01)。临床Ⅰ期和Ⅱ期鼻咽癌中Lactotransferrin蛋白的表达明显高于临床Ⅲ和Ⅳ期(p＜0.01)。提示它可能与鼻咽癌的侵袭转移和临床进展有关，为鼻咽癌候选易感基因，可作为鼻咽癌侵袭、转移和临床进展预测的分子靶标。
基因芯片和组织芯片结果均发现LTF基因在鼻咽癌中表达明显下调。为了明确LTF基因抑制鼻咽癌发生发展的可能作用机制，下一步拟探讨LTF基因对体外鼻咽癌细胞株的影响。
首先利用重组人乳铁传递蛋白(Recombinant human lactoferrin，rhlf)对鼻咽癌细胞株CNE1、5-8F、HNE1以及6-10B进行干预，MTT试验检测LTF基因对体外鼻咽癌细胞株生长的影响。结果发现LTF基因能够明显抑制鼻咽癌细胞CNE1、5-8F以及HNE1的生长。CNE1、5-8F以及HNE1细胞用10μmol／L rhlf干预后生长速度明显减慢(与未干预组比较)，干预24或48小时后即出现统计学差异。此结果再次从体外细胞角度验证了本论文第一部分的基因芯片和组织芯片结果。表明LTF不仅在鼻咽癌组织中表达明显下调，而且能够延缓体外鼻咽癌细胞株生长、抑制肿瘤形成。
然后，利用10μmol／L的rhlf对鼻咽癌细胞株ENE1、5-8F、HNE1以及6-10B进行干预，通过流式细胞术检测分析LTF基因对体外鼻咽癌细胞株细胞周期的影响。结果发现CNE1、5-8F以及HNEl细胞经10μmol／L的rhlf干预24或48小时后GO-G1期细胞百分比明显增多；S期和G2-M期细胞百分比则减少。但10μmol／L的rhlf干预对鼻咽癌6-10B细胞未产生明显影响。6-10B细胞是四种鼻咽癌细胞株中唯一一株没有转移潜能的，rhlf干预对其生长未产生明显的影响，这与前述基于组织微阵列(TMA)的免疫组织化学结果(LTF基因可能与鼻咽癌的侵袭转移和临床进展有关)互相印证，结果再次提示LTF主要作用于鼻咽癌发生发展的中晚期、在鼻咽癌转移过程中应该具有重要作用。此结果与MTT试验结果基本一致，证实LTF基因能够明显增加G0-G1期细胞比例、抑制体外鼻咽癌细胞的生长、增殖。
为了探讨LTF基因延缓G0-G1期进程、抑制鼻咽癌细胞生长的可能机制，采用具有高转移特性的鼻咽癌5-8F细胞作为研究对象，应用rhlf对其进行干预，利用western blot技术检测Cyclin D1，p-Rb，p21，p27，p53，总stat3，p-star3(Tyr705)以及MAPK信号传导通路中JNK2，c-Jun，总ERK1／2，p-ERK1／2，c-fos等相关信号分子干预前后的表达情况，分析LTF基因抑制体外鼻咽癌细胞增殖的作用机制。
鼻咽癌5-8F细胞用10μmol／L rhlf分别干预0min，5 min，10 min，30 min，60 min，12h，24h，36h，48h和72h后，抽提蛋白，western blot检测细胞周期蛋白cyclin D1、p-Rb的表达情况，结果发现：5-8F细胞用10μmol／L rhlf干预后周期蛋白cycl in D1、p-Rb的表达均低于对照组，说明LTF基因对周期蛋白cyclin D1、p-Rb的表达有重要影响。在此基础上，又检测了两种细胞周期素激酶(CDK)抑制蛋白p21，p27的表达情况。结果显示：鼻咽癌5-8F细胞用10μmol／L rhlf分别干预5 min，10 min，30 min，60 min，12h，24h，36h，48h和72h后，细胞周期素激酶(CDK)抑制蛋白p21，p27的表达与对照组相比(即0min)均增加。上述结果提示LTF基因通过调节周期蛋白cyclin D1、p-Rb以及细胞周期素激酶的表达来影响鼻咽癌5-8F细胞的增殖。
为了进一步了解引起cyclin D1、p-Rb改变的相关分子事件，根据已有试验结果、结合文献资料，我们选择MAPK信号传导通路的信号分子用western blot方法对其表达情况进行检测。鼻咽癌5-8F细胞用10μmol／Lrhlf分别干预5 min，10 min，30 min，60 min，12h，24h，36h，48h和72h后，抽提蛋白，western blot检测JNK2，c-Jun，总ERK1／2，p-ERK1／2和c-fos的表达情况。结果显示：LTF对总ERK1／2的表达未见明显影响；但降低p-ERK1／2的表达，用10μmol／L rhlf干预60min后其表达量明显减少，随着干预时间的延长，其表达量有进一步减少的趋势：LTF对JNK2蛋白表达产生影响，用10μmol／L rhlf干预12h后JNK2的表达量明显减少；同时LTF降低c-Jun的表达，用10μmol／L rhlf干预30min后即出现明显减少，随后就维持在一个较为平稳的水平；此外LTF对c-fos的表达有着较大影响，用10μmol／L rhlf干预60min后其表达量明显减少，随着干预时间的延长，其表达量有进一步减少的趋势，这种变化与p-ERR1／2的表达变动情况相吻合。
综上所述，LTF基因通过负性调控MAPK信号传导通路的JNK2和ERR亚家族，下调细胞周期蛋白cyclin D1，p-Rb以及细胞周期素激酶的表达，延缓鼻咽癌细胞G0-G1期进程，最终通过调节cyclins／CDKs／CKI／pRb为核心的G1／S期细胞周期网络调控系统来调控鼻咽癌5-8F细胞的生长、增殖。当然，基因表达调控系统是非常复杂和有机联系的，不同分子以及不同通路之间存在cross—talk，要明确LTF基因在其中所起的真正作用，还需以后进一步的实验证实。
【Identification of LTF Gene on the Region of NPC Susceptibility Gene—Chromosome 3p21】
In our previous study, linkage analysis of 18 pedigrees from Hunan Province of China suggests that potential susceptibility loci linked to NPC are located on chromosome 3p21. The putative NPC susceptibility locus is located a region from D3S1289 to D3S1298 on chromosome 3p21 through haplotype analysis. These results show that the candidate NPC susceptibility genes may be on chromosome 3p21.
To scan and identify the candidate NPC susceptibility genes on chromosome 3p21 further, two different gene chips were manufactured. One was cDNA microarray of subtracted cDNA libraries constructed by suppression subtractive hybridization, the other was a custom-made chromosome 3p21-specific cDNA microarray. Two different cDNA microarray were used to scran the same samples for improving their reliability.
First, the colonies used to made cDNA microarray were prepared by PCR from 4 NPC's subtracted cDNA libraries (NNTDH12, TRP19 DHNE1, TNPC DNN119, and TNN119 DNPC). There were more than 1,200 colonies including 300 colonies from every library to produce cDNA microarray. The purified colonies were used to made cDNA microarray of NPC's subtracted cDNA libraries.
After 19 NPCs, 3 NPC derived cell lines, and 10 chronic inflammation of nasopharyngeal mucosa tissue samples hybridized the cDNA microarray, primary data collection and analysis were carrded out via Genepix Pro 3.0 (Axon Instruments). The area of the array with obvious blemish was manually flagged and excluded from subsequent analysis. To minimize artifacts arising from low expression values, only genes with raw intensity values for both Cy3 and Cy5 of＞200 counts were chosen for differential analysis. Then overall intensifies were normalized with a correction coefficient obtained using the ratios of these genes. One thousand and ninety-seven valid colonies were obtained.
For the above preprocessed hierarchical clustering, colonies for which＞85% of measurements among all the samples were selected for further analysis. Altogether 898 of the 1,097 colonies passed this variation filter for the initial, then mean-centered and normalized with respect to other colonies in the sample and corresponding colonies in other samples. First, this list of colonies was used in Genesis software (http://genome.tugraz.at) to perform a hierarchical clustering analysis using the Euclidean distance and average linkage clustering. The specimens were divided into two subtypes based on these colonies: the NPC groups versus the chronic inflammation of nasopharyngeal mucosa groups. Within the "NPC" branch of the dendrogram, 3 NPC_derived cell lines were clustered as a small branch of NPC. T01_S was the replicate sample of the T01_F sample, and they were clustered tightly.
Though chronic inflammation of nasopharyngeal mucosa tissue and nasopharyngeal carcinoma tissue samples could be distinguished by 898 colonies, we hoped to find the smallest colony number to reach the aim. Genesis software was used for the following in ANOVA analysis to obtain the differentially expressed colonies among chronic inflammation of nasopharyngeal mueosa, NPCs and NPC-dedved cell lines. We obtained 105 differentially expression colonies. At last, these differentially expressed colonies were used in Genesis software to perform a hierarchical clustering analysis using the Euclidean distance and average linkage clustering. The specimens were divided into two subtypes based on these colonies: the NPC groups versus the chronic inflammation of nasopharyngeal mueosa groups. Within the "NPC" branch of the dendrogram, 3 NPC_derived cell lines were clustered as a small branch of NPC. T01_S was the replicate sample of the T01_F sample, which was clustered tightly. It showed that 105 colonies had the same test efficiency.
To identify the 105 colonies differentially expressed in NPC, all the cDNA colonies were amplified by using of the SSH secondary primers. The PCR products were purified from the gel anddirectly sequenced. The cDNA sequences were compared to GenBank by Blast search (http://www.nebi.nlm.nih.gov/BLAST/). There were 14 known genes, including STAT5A, RAB25, SPARC, and SPRR3, et al. to be identified in all the 37 colonies expressed highly in NPC and NPC_derived cell lines. Different frequency exists in every gene. STAT5A gene had the highest frequency of 18.92%, the second was RAB25(13.51%), followed by SPARC, SPRR3 and so on. These 14 genes were distributed on 9 chromosomes, including 1q, 2q, 4p, 5q, 7p, 7q, 12p, 12q, 13q, 15q and 17q. Eighteen known genes, including LTF, PLUNC, CDC37L1, HNRPDL genes, et al. were identified in 68 colonies which expressed poorly in N-PC and NPC_derived cell lines. The frequency to every gene was different. PLUNC gene had the highest frequency of 30.88%, the second was CDC37L1 (13.24%), followed by HNRPDL and so on. These 18 genes were distributed on 12 chromosomes, including lq, 3p, 4q, 7q, 8q, 9p, 11q, 12p, 12q, 14q, 16p, 20q and 22q. We scanned 32 genes differentially expressed in NPC using the cDNA mieroarray of subtracted cDNA libraries constructed by SSH, including LTF gene on the region of NPC susceptibility gene—ehromosome 3p21.
In addition, the results showed that lactotransferrin (LTF), dystroglyean 1, FLJ34969, and TU3A genes were down-regulated in NPC samples, glutamate receptor, metabotropie 2(GRM2) and interleukin 17 receptor B (IL17RB) were up-regulated in NPC samples by a custom-made chromosome 3p21-specifie cDNA mieroarray including 288 genes in this region in the same samples.
Through eompositive analysis of these results, we found that LTF was down-regulated in NPC samples in the two different gene chips. There was the parallel change in the same samples by different cDNA mieroarray. So we could believe that LTF gene might play an important role in pathogenesis of NPC.
To confirm the cDNA mieroarray results, we chose the same samples of cDNA mieroarray to perform real-time quantitative RT-PCR of LTF, PLUNC, CDC37L1, and HNRPDL genes, which were down-regulated in NPC samples, and the same was done to STAT5A, RAB25, and SPARC genes, which were up-regulated in NPC samples. The expression of LTF, PLUNC, CDC37L1, and HNRPDL genes were low in NPC and the expression of STAT5A, RAB25 and SPARC genes were high in NPC. It corresponded with the results of cDNA mieroarray and proved its reliability.
To validate the genes differentially expressed in NPC by cDNA mieroarray, we used in situ hybridization technique to confirm once more the expression of PLUNC, CDC37L1 and STAT5A genes with two NPC-tissue arrays. The positive expression rate of LTF and PLUNC genes in the NPC adjacent epitheliums and chromo nasopharyngeal was significantly higher than that of NPCs (p＜0.01). The positive expression rate of STAT5A gene in the NPC adjacent epitheliums and chromo nasopharyngeal was significantly lower than that of NPCs (p＜0.01). It corresponded with the results of cDNA microaray and proved its reliability.
【Function Study of LTF Gene that Inhibited Proliferation in the Nasopharyngeal Carcinoma Cells】
LTF was the parallel change in the different cDNA mieroarray. With compositive analysis of it and the previous results by linkage analysis of pedigrees, we had reasons to believe that LTF gene might play an important role in pathogenesis of NPC. At the same time, the expression of lactotransferrin protein was studied using immunoehemisty in NPC-tissue microarray by Dr. Zhao-yang ZENG. The results showed that laetotransferrin protein was down-regulated in NPC and up-regulated in NPC adjacent epitheliums and chronic inflammation of nasopharyngeal mueosas. Laetotransferrin protein could be detected in almost all of gland in nasopharynx, confirming that lactotransferrin protein was a secretory protein up-regulated in nasopharynx. In addition, the expression of laetotransferfin protein was negative correlated to the neck metastasis states (p＜0.05) and was significantly correlated to the clinical stages of NPC (p＜0.01). The expression of lactotransferrin protein in clinical stageⅠandⅡis obviously higher than that of the clinical stageⅢandⅣ. These findings suggest that LIF may be correlated with invading, metastasis and clinical evlovement and it is a good candidate tumor suppressor gene in NPC, and it may be a molecular marker for prediction of NPC invading, metastasis and clinical evlovement.
The results of cDNA mieroarray and TMA showed that LTF gene was down-regulated in NPC samples. To investigate the mechanism of LTF to NPC, we investigated the effects of LTF in the Nasopharyngeal Carcinoma Cells in vitro.
First, we used recombinant human laetoferrin (rhlf) to treat the NPC cell lines: CNE1, 5-8F, HNE1, and 6-10B. Then, we chose the reduction of the yellow tetrazolium salt3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay to assessment of proliferation- suppressing effect of LIF in NPC cell lines. The results of MTT assay showed that LTF significantly inhibited 5-8F, CNE1, and HNE1 cell proliferation after treated with lactoferrin. The growth of 5-8F, CNE1, and HNE1 cell treated with laetoferrin was suppressive compared with the control group. It was significantly different after treated with laetoferrin for 24 hours and 48 hours. It validated the results of cDNA microarray and TMA in part I from NPC cell lines in vitro. These findings suggested that LTF could reduce the growth of NPC cell lines in vitro and suppress the development of tumor, in addition, LTF was down-regulated in NPC samples.
To follow, we chose 10μmol/L recombinant human lactoferrin (rhlf) to treat the NPC cell lines: CNE1, 5-8F, HNE1, and 6-10B. Then, the experiment of flow cytometry was applied for cell cycle analysis of NPC cell lines to evaluate the effect of LTF. The findings showed that the percentage of G0-G1 phase of the cell cycle in 5-8F, CNE1, and HNE1 cells was significantly increased after treated with lactoferrin (10μmol/L) for 24 hours or 48 hours, but the percent of G2-M phase and S phase was reduced respectively. But it had no significantly effect to 6-10B cell after treated with lactoferrin. Among 5-8F, CNE1, HNE1, and 6-10B cells, 6-10B cell was the only one that had no characteristic of metastasis. It corresponded with the results of immunochemisty in NPC-tissue microarray- LTF might be correlated with invading, metastasis and clinical evolvement. The results hinted again that LTF mainly functioned in the mid-terminal stages of NPC and played an important role in the stages. These data were consistent with results of MTT army. Moreover, it proved that LTF could suppress the growth and proliferation of NPC cells in vitro.
To reveal underlying mechanism that LTF delayed G0-G1 phase progress to inhibit growth of NPC cells in vitro, we chose 5-8F cell line with high characteristic of metastasis as investigation object. After 5-8F cells were treated with lactoferrin, we examined the expression levels of Cyclin D1, p-Rb, p21, p27, p53, total star3, p-stat3(Tyr705) and signal molecules of mitogen-activated protein kinase (MAPK) signal pathway including JNK2, c-Jun, total ERK1/2, p-ERK1/2, and c-fos by western-blot technique. At last, we hoped to reveal some underlying mechanism that LTF inhibited the growth and proliferation of NPC cells in vitro.
After NPC derived cell lines 5-8F was treated with lactoferrin (10μmol/L) for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, we prepared their protein in turn and tested the expression levels of cyclin D1 and phosphorylated Rb by western blot. After treated with lactoferrin, the expression levels of cyclin D1 and phosphorylated Rb were reduced distinctly compared with the corresponding levels in the untreated. The results suggested that LTF affected the expression of cyclin D1 and p-Rb. Subsequently, we studied the expression of p21 and p27 proteins that were cyclin-dependent kinase inhibitor. After treated with lactoferrin for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, the expression levels of p21 and p27 proteins were increased significantly compared with the corresponding levels in the untreated. In conclusion, these findings suggested that LTF could suppress the proliferation of NPC derived cell lines 5-8F through regulating the moeulars of cyclin D1, p-Rb, cyclin-dependent kinase, and cyclin-dependent kinase inhibitor.
To additionally realize the molecular changes associated with cyclin D1 and p-Rb, we examined the signal molecules of mitogen-activated protein kinase (MAPK) signal pathway by western blot according to our previous results and accumulated document evidences. After NPC derived cell lines 5-8F was treated with lactoferrin (10μmol/L) for 0min, 5min, 10min, 30min, 60min, 12h, 24h, 36h, 48h, and 72h respectively, we prepared their protein in turn and tested the expression levels of JNK2, c-Jun, total ERK1/2, p-ERK1/2, and c-fos by western-blot technique. The expression level of p-ERK1/2 was reduced significantly after treated with lactoferrin (10μmol/L) for 60min. There was a decreasing trend of expression quantity with extension of treatment time. But we found that LTF gene had no effect on the expression level of total ERK. The expression level of JNK2 was reduced obviously after treated with lactoferrin (10μmol/L) for 12h. LTF gene affected the expression level of JNK2 protein. LTF gene cut down the expression levels of not only JNK2 protein but also c-Jun protein. The expression level of c-Jun was decreased obviously after treated with lactoferrin (10μmol/L) for 30min, and then was kept at an invariablenes level. In addition, LTF gene had an important effect on the expression of c-fos. The expression level of c-fos was reduced significantly after treated with lactoferrin (10μmol/L) for 60min. There was a decreasing trend of expression quantity with extension of treatment time. It corresponded with the changes ofp-ERK1/2.
In summary, LTF negatively modulated sub-families of JNK2, and ERK of MAPK signal pathway to suppress the expression of cyelin D1, p-Rb, and Cyelin-Dependent Kinase and to delay the G0-G1 progression of nasopharyngeal carcinoma cells. LTF inhibited growth and proliferation of 5-8F cells in vitro by modulating the G1/S phase regulation systems with regulating eyelins/CDKs/CKI/pRb as the key point. Subsequently, regulation systems of gene expression are very complicated and organic contacted. There are some cross-talks among different pathways anddifferent moleeulars. To reveal the reality ftmetions of LTF, we must do more experiments to verify it farther.
引文
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14 Yin J, Chen MF, Finkel TH.Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.AIDS, 2004,18(4):587-596.
15 Zhang YL,Ong CT,Leung KY. Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology,2000,146:999-1009.
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1 Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992, 257: 967-971.
2 Hwang KC, Park SY, Park SP, et al. Specific maternal transcripts in bovine oocytes and cleavaged embryos: identification with novel DDRT-PCR methods. Mol Reprod Dev, 2005, 71(3): 275-283.
3 Lang P, Zhang CK, Ebel RC, et al. Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display. Gene, 2005, 359: 111-118.
4 Fauchon MA, Pell TJ, Baxter GF, et al. Representational difference analysis of cDNA identifies novel genes expressed following preconditioning of the heart. Exp Mol Med, 2005 ,37(4):311-322.
5 Oue N, Aung PP, Mitani Y,et al. Genes involved in invasion and metastasis of gastric cancer identified by array-based hybridization and serial analysis of gene expression.Oncology, 2005;69(Suppl 1): 17-22.
6 Derisi J,Penland L,Brown PO,et al.Use of a cDNA microarray to analyse gene expression patterns in human cancer.Net Genet, 1996,14(4):457-460.
7 Diatchenko L,Lau YF,Campbell AP,et al. Suppression subtractive hybridization: a method for generating differentially regulating or tissue-specific cDNA probes and libraries.Proc Natl Acad Sci USA,1996,93:6025-6030.
8 Sun G, Liu F, Lin TJ. Identification of Pseudomonas aeruginosa-induced genes in human mast cells using suppression subtractive hybridization: up-regulation of IL-8 and CCL4 production.CIin Exp Immunol, 2005 ,142(1):199-205.
9 Schena M,Dhalon D,Davis RW,et al.Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995,270:467-470.
10 Shou J, Dotson C, Qian HR,et al.Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray.Biomarkers, 2005;10(4):310-320.
11 Sun W, Zhang K, Zhang X,et al.Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.Cancer Lett, 2004,212(1):83-93.
12 Schulz HL, Rahman FA, Fadl El Moula FM,et al.Identifying differentially expressed genes in the mammalian retina and the retinal pigment epithelium by suppression subtractive hybridization.Cytogenet Genome Res, 2004; 106(1):74-81.
13 Shimada T,Kawai T,Takeda K,et al. IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IkappaB kinases. Int Immunol, 1999,11(8): 1357-1362.
14 Yin J, Chen MF, Finkel TH.Differential gene expression during HIV-1 infection analyzed by suppression subtractive hybridization.AIDS, 2004,18(4):587-596.
15 Zhang YL,Ong CT,Leung KY. Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish. Microbiology,2000,146:999-1009.
16 Duggan DJ, Bittner M, Chen Y,et al. Expression profiling using DNA microarrays. Nature Genetics (Supplement) ,1999;21; 10-14
17 Schena M ,Shalon D,Davis R,et al.Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Science,1995,270(5235):467-470.
18 Zhou YH, Zeng ZY, Xiong W, et al. Microdissection and RNA line-amplification of Nasopharyngeal Carcinoma Tissue. Prog Biochem Biophys,2005; 32(5):463-467.
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