PLUNC基因启动子区SNP位点C-1888T与鼻咽癌易感性的转录调控研究

发布时间：2018-06-27 10:41

本文选题：PLUNC基因 + 鼻咽肿瘤　；参考：《南方医科大学》2011年硕士论文

【摘要】：研究背景和目的鼻咽癌(nasopharyngeal carcinoma, NPC)是我国南方及东南亚地区常见的一类恶性肿瘤,目前认为EB病毒感染、化学促癌和/或致癌物等在鼻咽癌致瘤过程中均可能起到重要作用。鼻咽癌的发生同其他肿瘤一样,是一个多因素参与和多阶段的过程。为了研究鼻咽癌发病过程中的基因表达变化,姚开泰院士、何志巍博士等通过含有高密度的基因/表达序列标签(ESTs)的cDNA微阵列膜比较了人正常鼻咽和鼻咽癌组织的基因差异表达谱,并从中克隆出一个具有组织相对特异性表达的候选抑瘤基因—YH1基因,GeneBank收录号为AF158745。YH1仅高表达于人的鼻咽和气管,具有明显的上皮特异性表达的特征,被认为是一个新的与鼻咽癌相关的基因,具有很好的组织特异性。随后其他课题组在小鼠胚胎的鼻咽上皮及成人肺、呼吸道和鼻咽上皮克隆出称为PLUNC(palate, lung and nasal epithelium clone, PLUNC)的新基因,同源性分析表明,YH1和PLUNC为同一序列,该基因在猪、牛、大鼠等物种中都高度保守,因而被统一归结为PLUNC家族。根据相关文献报导,位于基因编码区、侧翼区和调控区的单核苷酸多态性(Single Nucleotide Polymorphism, SNP)与相关疾病联系紧密,可能会改变基因功能。近年来,我们在PLUNC基因编码区、调控区和侧翼区分别设计出特异性引物,并从中发现9个SNPs位点。在获得该基因在中国人群样本中等位频率的SNP位点的信息后,对其中数个SNPs位点展开分型研究,发现PLUNC基因启动子区2个多态位点C-2128T和C-1888T与中国人群鼻咽癌易感性关系非常密切(OR=2.8-3.3,P0.001),在此基础上进行的单体型分类也显示出携带单体型C-C的个体更易患鼻咽癌(OR=1.86,95% CI=1.34-2.56,P=-0.00016)。荧光素酶活性结果检测示：1888 C启动子活性明显低于PLUNC基础启动子——1888 T的活性,且其活性显著下降了约64.67%,两者差异有统计学意义。上述结果提示我们C-1888T位点多态性可能影响了该基因的转录调控,与鼻咽癌的易感／风险相关。基于上述研究背景,我们认为有必要对PLUNC基因启动子区进行深入研究,验证并加强前期结果的可靠性；同时对启动子区多态位点C-1888T开展初步功能性研究,验证该多态位点是否影响了该基因的转录调控并与鼻咽癌的易感／风险相关。方法 1、采用聚合酶链式反应-测序方法及聚合酶链式反应-限制性片段长度多态性方法(polymerase chain reaction-Restriction fragment length polymorphism, PCR-RFLP),从已建株的鼻咽癌细胞(CNE-1、CNE-2、SUNE-1、5-8F、6-10B、C666-1)及鼻咽癌患者鼻咽病变组织原代培养细胞中筛选PLUNC基因启动子区C-1888T SNP位点三种基因型。 2、提取上述各已建株的鼻咽癌细胞中的总RNA,逆转录成cDNA,内参采用β-actin,使用荧光定量PCR仪Mx3005P进行实时定量PCR扩增,检测不同细胞株中PLUNC的相对表达量。结果采用One-Way ANOVA统计学方法分析。 3、运用p-match (www.gene-regulation.com/pub/programs.html#pmatch)、Consite (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite/)等软件分析,当C-1888T SNP位点分别为A或G时,与之结合可能性较大的转录因子,通过TRANSFAC数据库所给出的结合位点矩阵信息,得到不同转录因子结合的核心序列,并设计相应的探针和突变体探针。 4、提取对数生长期的CC和TT型细胞的核蛋白,BCA法测定蛋白浓度,进行寡核苷酸探针的生物素标记。将核蛋白与生物素标记的探针进行结合反应,同时设置不加核蛋白的阴性对照组、特异性竞争抑制组和非特异性竞争抑制组,检测泳道阻滞条带。 5、进一步应用染色质免疫共沉淀技术结合PCR扩增,对CC型鼻咽癌细胞株进行分析,以确定转录因子EVI1可与PLUNC基因启动子区特异结合。结果 1、5-8F、6-10B、CNE1、CNE2为杂合子CT型,SUNE1为纯合子CC型,C666-1为纯合子TT型。PCR特异性扩增产物直接测序,其结果与PCR-RFLP判读分析比较,完全一致。生长状态良好的原代培养细胞均为CT型,CC及TT型原代培养细胞生长状态欠佳甚至死亡,无法满足后继实验需要。 2、PLUNC基因在不同细胞株中的表达存在显著差异(F=33.844,P=0.000)；此外,LSD法多重比较发现,CC型与TT型细胞株中基因表达也存在显著差异,CC型细胞中基因表达水平明显比TT型低(P=0.000)。 3、相关软件分析结果显示：XFD3和EVI1分别在C-1888T位SNP位点为A和G时,有很大的结合可能性(score分别达0.96和1.00)。其结合的核心序列分别为TTGGTCAACAAGAT和CGACAAGATAA。后继实验也证实所选择的软件分析预测结果准确可靠。 4、PLUNC基因SNP位点C-1888T分别为A和G时,探针分别可以和转录因子XFD3及EVI1结合形成阻滞条带,并可被特异性竞争抑制,而突变体探针却不能抑制二者的结合。 5、以EVI1抗体免疫沉淀的染色质片段提取的CC型细胞DNA为模板,PCR扩增后显示：Anti-RNA Polymerase IIChIP DNA、EVI1 antibody ChIP DNA、InputDNA扩增后在192bp处出现条带,而阴性对照IgG ChIP DNA扩增后并未出现条带。结论 1、体外稳定建株的鼻咽癌细胞启动子区同样存在多态位点C-1888T,与人类基因数据库一致,可用于进行相关的体外研究。 2、CC型细胞中基因表达水平明显比TT型低(P=0.000),结合我们课题组前期结果：1888 C启动子活性明显低于PLUNC基础启动子——1888 T的活性(活性显著下降了约64.67%,两者差异有统计学意义),说明C-C型个体更易患鼻咽癌可能是通过下调了PLUNC基因的表达而实现。 3、生物信息学预测是基因启动子区转录因子结合部位分析必不可少的工具之一,具有一定指导作用,本实验前期预测结果与后期实验结果相符,证实预测的准确性。 4、EMSA实验初步证实两个转录因子XFD3和EVI1与DNA的结合是特异的,它们可能参与了PLUNC基因的转录调控,影响了PLUNC基因的表达。 5、ChIP实验进一步验证转录因子EVI1可以和CC型鼻咽癌细胞PLUNC基因启动子区结合,参与PLUNC基因的转录调控。
[Abstract]:Background and purpose of research
Nasopharyngeal carcinoma (NPC) is a common type of malignant tumor in South and Southeast Asia. It is considered that EB virus infection, chemical cancer promoting and / or carcinogen may play an important role in the carcinogenesis of nasopharyngeal carcinoma. The occurrence of nasopharyngeal carcinoma, like other swollen tumors, is a multi factor participation and multi stage process. In order to study the changes in gene expression during the pathogenesis of nasopharyngeal carcinoma, academician Yao Kaitai and Dr. He Zhiwei compared the gene differential expression profiles of human normal nasopharyngeal and nasopharyngeal carcinoma tissues by using a cDNA microarray containing high density gene / expression sequence tag (ESTs), and cloned a relatively specific expression of tissue in the tissues of human nasopharynx and nasopharynx. The tumor suppressor gene YH1 gene, which is only highly expressed in the human nasopharynx and trachea, is highly expressed in the human nasopharynx and trachea. It is considered to be a new gene related to nasopharyngeal carcinoma and has a good tissue specificity. Then, the other subjects are in the nasopharyngeal epithelium and adult lung and respiratory tract of the mouse embryos. A new gene called PLUNC (palate, lung and nasal epithelium clone, PLUNC) was cloned from the nasopharyngeal epithelium. The homology analysis showed that YH1 and PLUNC were the same sequence. The gene was highly conserved in pigs, cattle and rats, and was unified as a PLUNC family.
According to the relevant literature, the single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNP) in the gene coding region, the flanking region and the regulatory region are closely related to the related diseases, which may change the gene function. In recent years, we have designed specific primers in the PLUNC gene coding region, the regulatory area and the flanking region, and found 9 SNP from the gene coding region. S loci. After obtaining the information of the SNP locus at the medium frequency of the Chinese population, several SNPs loci were unfolded. The 2 polymorphic loci of the PLUNC gene promoter region C-2128T and C-1888T were found to be closely related to the susceptibility to nasopharyngeal carcinoma (OR= 2.8-3.3, P0.001) in Chinese population (OR= 2.8-3.3, P0.001), based on the haplotype. The taxonomy also showed that individuals with haplotype C-C were more susceptible to nasopharyngeal carcinoma (OR=1.86,95% CI=1.34-2.56, P=-0.00016). The results of luciferase activity showed that the activity of 1888 C promoter was significantly lower than that of PLUNC base promoter, 1888 T, and its activity decreased by about 64.67%, and the difference between them was statistically significant. The above results suggested that the difference was statistically significant. The polymorphism of our C-1888T locus may affect the transcriptional regulation of this gene, which is associated with susceptibility / risk of nasopharyngeal carcinoma.
Based on the above research background, we think it is necessary to study the promoter region of the PLUNC gene in depth to verify and strengthen the reliability of the early results. At the same time, a preliminary functional study of the polymorphic loci C-1888T in the promoter region is carried out to verify whether the polymorphic locus affects the gene regulation and the susceptibility / risk phase of nasopharyngeal carcinoma. Close.
Method
1, by using polymerase chain reaction sequencing and polymerase chain reaction restriction fragment length polymorphism (polymerase chain reaction-Restriction fragment length polymorphism, PCR-RFLP), nasopharyngeal carcinoma cells (CNE-1, CNE-2, SUNE-1,5-8F, 6-10B, C666-1) and nasopharyngeal carcinoma were cultured for primary culture of nasopharyngeal carcinoma. The cells screened three genotypes of PLUNC gene promoter region C-1888T SNP locus.
2, the total RNA of the nasopharyngeal carcinoma cells were extracted, and cDNA was reverse transcriptase. The internal reference was used to detect the relative expression of PLUNC in different cell lines by using the fluorescence quantitative PCR Mx3005P to detect the relative expression of PLUNC in different cell lines. The results were analyzed by the One-Way ANOVA statistical method.
3, using p-match (www.gene-regulation.com/pub/programs.html#pmatch), Consite (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite/) and other software analysis, when the C-1888T SNP loci are A or G, they are combined with the larger possibility of transcription factors, and get different transfers through the information of the binding site matrix given by the TRANSFAC database. The core sequences are combined with the corresponding probes and probes.
4, the nucleoprotein of CC and TT cells in the logarithmic growth period was extracted, the protein concentration was measured by BCA, and the biotin labeling of the oligonucleotide probe was carried out. The nucleoprotein and biotin probe were combined, and the negative control group, the specific competition suppressor group and the non specific competition inhibition group, were set up, and the lane resistance was detected. Sluggish strip.
5, the chromatin immunoprecipitation technique combined with PCR amplification was used to analyze the CC type nasopharyngeal carcinoma cell lines to determine the specific binding of the transcription factor EVI1 to the promoter region of the PLUNC gene.
Result
1,5-8F, 6-10B, CNE1, CNE2 are heterozygote CT, SUNE1 is homozygote CC, C666-1 is a homozygote TT type.PCR specific amplification product direct sequencing. The results are compared with PCR-RFLP interpretation analysis. The successor experiment needs.
2, the expression of PLUNC gene in different cell lines was significantly different (F=33.844, P=0.000). In addition, multiple comparison of LSD method found that there was significant difference in gene expression in CC and TT cell lines, and the level of gene expression in CC cells was significantly lower than that of TT type (P=0.000).
3, the results of the related software analysis show that XFD3 and EVI1 have a great possibility of binding at the C-1888T site SNP loci of A and G respectively (score is 0.96 and 1 respectively). The core sequence of the combination is TTGGTCAACAAGAT and CGACAAGATAA. succeeding experiments, respectively, which also confirmed that the selected software analysis prediction results are accurate and reliable.
4, when the SNP locus C-1888T of the PLUNC gene is A and G, the probe can be combined with the transcription factor XFD3 and EVI1 to form a block band, which can be inhibited by specific competition, while the mutant probe can not inhibit the combination of the two.
5, the CC cell DNA extracted from the chromatin fragment of EVI1 antibody immunoprecipitation was the template, and PCR amplification showed that Anti-RNA Polymerase IIChIP DNA and EVI1 antibody ChIP DNA.
conclusion
1, the stable loci of nasopharyngeal carcinoma cells in the promoter region also have polymorphic loci C-1888T, which is consistent with the human genome database and can be used for relevant in vitro studies.
2, the gene expression level in CC type cells was significantly lower than that of TT type (P=0.000). Combined with the previous results of our group, the activity of 1888 C promoter was significantly lower than that of PLUNC base promoter, 1888 T (the activity was significantly decreased by about 64.67%, the difference was statistically significant), suggesting that C-C type individuals were more likely to suffer from nasopharyngeal carcinoma by downregulating PLUN The expression of C gene is realized.
3, bioinformatics prediction is one of the indispensable tools for the analysis of the binding site of the gene promoter region, which has a certain guiding role. The prediction results in the early stage of this experiment coincide with the experimental results, confirming the accuracy of the prediction.
4, the EMSA experiment preliminarily confirmed that the combination of two transcription factors, XFD3 and EVI1, and DNA are specific. They may be involved in the transcription regulation of the PLUNC gene and affect the expression of the PLUNC gene.
5, the ChIP experiment further verified that transcription factor EVI1 could bind to the PLUNC gene promoter region of CC nasopharyngeal carcinoma cell and participate in the transcriptional regulation of PLUNC gene.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

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