本文選題：PLUNC基因 + 鼻咽腫瘤��； 參考：《南方醫(yī)科大學》2011年碩士論文

【摘要】：研究背景和目的 鼻咽癌(nasopharyngeal carcinoma, NPC)是我國南方及東南亞地區(qū)常見的一類惡性腫瘤,目前認為EB病毒感染、化學促癌和/或致癌物等在鼻咽癌致瘤過程中均可能起到重要作用。鼻咽癌的發(fā)生同其他腫瘤一樣,是一個多因素參與和多階段的過程。為了研究鼻咽癌發(fā)病過程中的基因表達變化,姚開泰院士、何志巍博士等通過含有高密度的基因/表達序列標簽(ESTs)的cDNA微陣列膜比較了人正常鼻咽和鼻咽癌組織的基因差異表達譜,并從中克隆出一個具有組織相對特異性表達的候選抑瘤基因—YH1基因,GeneBank收錄號為AF158745。YH1僅高表達于人的鼻咽和氣管,具有明顯的上皮特異性表達的特征,被認為是一個新的與鼻咽癌相關的基因,具有很好的組織特異性。隨后其他課題組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稱為PLUNC(palate, lung and nasal epithelium clone, PLUNC)的新基因,同源性分析表明,YH1和PLUNC為同一序列,該基因在豬、牛、大鼠等物種中都高度保守,因而被統(tǒng)一歸結(jié)為PLUNC家族。 根據(jù)相關文獻報導,位于基因編碼區(qū)、側(cè)翼區(qū)和調(diào)控區(qū)的單核苷酸多態(tài)性(Single Nucleotide Polymorphism, SNP)與相關疾病聯(lián)系緊密,可能會改變基因功能。近年來,我們在PLUNC基因編碼區(qū)、調(diào)控區(qū)和側(cè)翼區(qū)分別設計出特異性引物,并從中發(fā)現(xiàn)9個SNPs位點。在獲得該基因在中國人群樣本中等位頻率的SNP位點的信息后,對其中數(shù)個SNPs位點展開分型研究,發(fā)現(xiàn)PLUNC基因啟動子區(qū)2個多態(tài)位點C-2128T和C-1888T與中國人群鼻咽癌易感性關系非常密切(OR=2.8-3.3,P0.001),在此基礎上進行的單體型分類也顯示出攜帶單體型C-C的個體更易患鼻咽癌(OR=1.86,95% CI=1.34-2.56,P=-0.00016)。熒光素酶活性結(jié)果檢測示：1888 C啟動子活性明顯低于PLUNC基礎啟動子——1888 T的活性,且其活性顯著下降了約64.67%,兩者差異有統(tǒng)計學意義。上述結(jié)果提示我們C-1888T位點多態(tài)性可能影響了該基因的轉(zhuǎn)錄調(diào)控,與鼻咽癌的易感／風險相關。 基于上述研究背景,我們認為有必要對PLUNC基因啟動子區(qū)進行深入研究,驗證并加強前期結(jié)果的可靠性；同時對啟動子區(qū)多態(tài)位點C-1888T開展初步功能性研究,驗證該多態(tài)位點是否影響了該基因的轉(zhuǎn)錄調(diào)控并與鼻咽癌的易感／風險相關。 方法 1、采用聚合酶鏈式反應-測序方法及聚合酶鏈式反應-限制性片段長度多態(tài)性方法(polymerase chain reaction-Restriction fragment length polymorphism, PCR-RFLP),從已建株的鼻咽癌細胞(CNE-1、CNE-2、SUNE-1、5-8F、6-10B、C666-1)及鼻咽癌患者鼻咽病變組織原代培養(yǎng)細胞中篩選PLUNC基因啟動子區(qū)C-1888T SNP位點三種基因型。 2、提取上述各已建株的鼻咽癌細胞中的總RNA,逆轉(zhuǎn)錄成cDNA,內(nèi)參采用β-actin,使用熒光定量PCR儀Mx3005P進行實時定量PCR擴增,檢測不同細胞株中PLUNC的相對表達量。結(jié)果采用One-Way ANOVA統(tǒng)計學方法分析。 3、運用p-match (www.gene-regulation.com/pub/programs.html#pmatch)、Consite (http://mordor.cgb.ki.se/cgi-bin/CONSITE/consite/)等軟件分析,當C-1888T SNP位點分別為A或G時,與之結(jié)合可能性較大的轉(zhuǎn)錄因子,通過TRANSFAC數(shù)據(jù)庫所給出的結(jié)合位點矩陣信息,得到不同轉(zhuǎn)錄因子結(jié)合的核心序列,并設計相應的探針和突變體探針。 4、提取對數(shù)生長期的CC和TT型細胞的核蛋白,BCA法測定蛋白濃度,進行寡核苷酸探針的生物素標記。將核蛋白與生物素標記的探針進行結(jié)合反應,同時設置不加核蛋白的陰性對照組、特異性競爭抑制組和非特異性競爭抑制組,檢測泳道阻滯條帶。 5、進一步應用染色質(zhì)免疫共沉淀技術結(jié)合PCR擴增,對CC型鼻咽癌細胞株進行分析,以確定轉(zhuǎn)錄因子EVI1可與PLUNC基因啟動子區(qū)特異結(jié)合。 結(jié)果 1、5-8F、6-10B、CNE1、CNE2為雜合子CT型,SUNE1為純合子CC型,C666-1為純合子TT型。PCR特異性擴增產(chǎn)物直接測序,其結(jié)果與PCR-RFLP判讀分析比較,完全一致。生長狀態(tài)良好的原代培養(yǎng)細胞均為CT型,CC及TT型原代培養(yǎng)細胞生長狀態(tài)欠佳甚至死亡,無法滿足后繼實驗需要。 2、PLUNC基因在不同細胞株中的表達存在顯著差異(F=33.844,P=0.000)；此外,LSD法多重比較發(fā)現(xiàn),CC型與TT型細胞株中基因表達也存在顯著差異,CC型細胞中基因表達水平明顯比TT型低(P=0.000)。 3、相關軟件分析結(jié)果顯示：XFD3和EVI1分別在C-1888T位SNP位點為A和G時,有很大的結(jié)合可能性(score分別達0.96和1.00)。其結(jié)合的核心序列分別為TTGGTCAACAAGAT和CGACAAGATAA。后繼實驗也證實所選擇的軟件分析預測結(jié)果準確可靠。 4、PLUNC基因SNP位點C-1888T分別為A和G時,探針分別可以和轉(zhuǎn)錄因子XFD3及EVI1結(jié)合形成阻滯條帶,并可被特異性競爭抑制,而突變體探針卻不能抑制二者的結(jié)合。 5、以EVI1抗體免疫沉淀的染色質(zhì)片段提取的CC型細胞DNA為模板,PCR擴增后顯示：Anti-RNA Polymerase IIChIP DNA、EVI1 antibody ChIP DNA、InputDNA擴增后在192bp處出現(xiàn)條帶,而陰性對照IgG ChIP DNA擴增后并未出現(xiàn)條帶。 結(jié)論 1、體外穩(wěn)定建株的鼻咽癌細胞啟動子區(qū)同樣存在多態(tài)位點C-1888T,與人類基因數(shù)據(jù)庫一致,可用于進行相關的體外研究。 2、CC型細胞中基因表達水平明顯比TT型低(P=0.000),結(jié)合我們課題組前期結(jié)果：1888 C啟動子活性明顯低于PLUNC基礎啟動子——1888 T的活性(活性顯著下降了約64.67%,兩者差異有統(tǒng)計學意義),說明C-C型個體更易患鼻咽癌可能是通過下調(diào)了PLUNC基因的表達而實現(xiàn)。 3、生物信息學預測是基因啟動子區(qū)轉(zhuǎn)錄因子結(jié)合部位分析必不可少的工具之一,具有一定指導作用,本實驗前期預測結(jié)果與后期實驗結(jié)果相符,證實預測的準確性。 4、EMSA實驗初步證實兩個轉(zhuǎn)錄因子XFD3和EVI1與DNA的結(jié)合是特異的,它們可能參與了PLUNC基因的轉(zhuǎn)錄調(diào)控,影響了PLUNC基因的表達。 5、ChIP實驗進一步驗證轉(zhuǎn)錄因子EVI1可以和CC型鼻咽癌細胞PLUNC基因啟動子區(qū)結(jié)合,參與PLUNC基因的轉(zhuǎn)錄調(diào)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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R739.63

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