【作者】 王爽；

【导师】 姚开泰；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 恶性肿瘤是严重危害人类健康的常见病、多发病。从本质上来说肿瘤是基因病，其发生、发展过程是多种基因表达失常的结果，因此真正意义上的肿瘤治疗也应该、同时也必须是以修正这些发生变化的基因为基点的治疗手段。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。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，何志巍博士通过高密度的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，并从中克隆了一个表达差异具有显著性的EST全长，命名为YH1基因，GeneBank收录号为AF158745。YH1基因定位在染色体20q11.2，基因全长约7.31kb，包括9个外显子和8个内含子；cDNA含有完整的阅读框架，编码一个富含亮氨酸（24.6％）、含256个氨基酸的偏酸、疏水性蛋白质，有4个磷酸化位点。由于其3’UTR剪切方式不同，存在2种不同的转录本，但编码的蛋白质相同。YH1基因序列与小鼠的上腭、肺及鼻咽上皮克隆（palate，lung and nasal epithelium clone，PLUNC）基因高度同源，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；因其分子量小，因此人PLUNC基因被命名为SPLUNC1（short plunc 1）。SPLUNC1基因的表达具有相对的组织特异性。在成人鼻咽组织中有较强的表达，而在鼻咽癌细胞系及活检组织中表达下调或不表达；气管组织中的表达水平明显高于右心房、空肠、成人肺、唾液腺、胚胎肺组织等。此外，SPLUNC1基因的一个转录本LUNX在非小细胞性肺癌中表达上调，而且能在该肿瘤的早期转移淋巴结中检测到，因而LUNX被认为是一个非小细胞性肺癌淋巴结早期转移的分子标志物。SPLUNC1基因确切的生物学功能至今仍不明确。生物信息学分析显示，该基因编码蛋白的N-端存在一个含19个氨基酸的信号肽，氨基酸序列与腮腺和气管腺上皮产生的分泌蛋白LCN1和腮腺分泌蛋白（parotid secretary protein）极为相似。对该蛋白三维结构的预测结果显示内部含有一个杀菌／通透性增强蛋白（bactericidal／permeability-increasing protein，BPI）结构域（BPI domain），其三维折叠结构与BPI折叠极为相似，而BPI的桶状结构能与细菌细胞壁上脂多糖特异性的结合，从而具有中和内毒素及直接杀菌作用，因此推测SPLUNC1蛋白很可能是一种分泌蛋白，并具有结合细菌脂多糖、消灭细菌的功能。此：外，对SPLUNC1基因的单核苷酸多态性的研究也证实该基因的启动子区多态性与NPC的易感性之间存在着联系，是NPC发生的一个风险因子。基因表达调控机制是后基因组时代一个重要的研究内容。基因的正确表达依赖于一个复杂的调控机制，主要发生在转录水平。启动子是与RNA聚合酶特异结合的DNA序列，是转录起始所必需的顺式元件。在基因表达的调控中，特定的启动子起始过程常常决定某个基因是否应当表达。增强子是基因转录的正调控顺式作用元件，具有促进启动子表达下游基因的作用，同其他的负调控元件和反式作用因子（转录因子）共同维护着下游基因的正确表达。不同的转录因子对外界环境的各种刺激或不同发育阶段的各种信号做出反应，结合于转录调控元件，激活或抑制基因的转录，从而控制不同基因的表达。一个基因的正确表达依赖于顺式作用元件、反式作用因子和RNA聚合酶三者之间的相互协同作用。在阐明基因转录调控机制的基础上，针对性地干预某些转录因子与DNA的结合，逆转基因异常表达，产生抗肿瘤效应，已经发展成为肿瘤治疗的新方法。基于人SPLUNC1基因的研究现状及其在肿瘤发病机制和防治中的潜在意义，本课题的研究目的是明确人体内表达SPLUNC1的细胞类型，鉴定SPLUNC1的启动子、增强子和有关的转录调控因子，为深入研究SPLUNC1基因和相关调控机制、构建组织特异性启动子及寻找新的治疗药物和作用靶点奠定基础。本课题进行了如下研究：（一）表达SPLUNC1基因的细胞类型的鉴定采用原位杂交方法检测近30种组织中SPLUNC1基因的表达情况，结果显示SPLUNC1在上腭、表皮、食管及食管—责门部的鳞状上皮，鼻咽低分化鳞状细胞癌、食管鳞癌和肺鳞癌的肿瘤细胞，鼻咽部、气管、宫颈部化生的鳞状上皮中均不表达；在胃粘膜、胆囊、空肠、结肠、子宫内膜及腺体和宫颈部的单层柱状上皮细胞中也不表达；主要表达在鼻咽、气管和支气管的假复层柱状上皮中，且沿呼吸道从上至下，该基因表达逐渐减弱，在肺组织中检测不到其表达。在腮腺、下颌下腺的导管和浆液腺细胞、鼻咽、肺和食管等部位的粘膜下浆液腺细胞、胃粘膜下壁细胞及乳腺小叶和导管上皮中也表达SPLUNC1，而粘液腺细胞中不表达。此外，在肺、胃、结肠、乳腺、子宫内膜和宫颈等部位的腺癌组织中，SPLUNC1基因均呈强阳性表达。提示SPLUNC1基因很可能是一个与分化有关的基因，而非鼻咽癌的抑癌基因，其表达具有明显的细胞特异性。（二）生物信息学预测SPLUNC1基因表达调控元件采用多个生物信息学软件对SPLUNC1基因5’端-5000bp～+1000bp（以第一外显子为+1位）序列进行分析，预测SPLUNC1的转录起始位点位于-1～+10bp区域间、-29bp处存在TATA box、启动子位于-490～+89bp区间；在人和小鼠两物种间该基因的-245～-1bp区间高度保守，并且存在多个转录因子结合位点。这些预测结果为进一步鉴定SPLUNC1基因的调控元件、探讨其表达调控机制和构建组织特异性启动子奠定了基础，具有一定的参考价值。（三）SPLUNC1基因5’端侧翼区启动子活性分析通过Real-time RT-PCR的方法检测了肺腺癌等十一种细胞系中SPLUNC1基因的表达水平，结果显示在肺腺癌Glc-82细胞中该基因的表达水平最高，这为进一步研究SPLUNC1基因的调控机制提供了合适的细胞材料。在生物信息学预测结果的基础上，以人基因组DNA为模板，采用PCR方法扩增了5’端截短的P4（-3000～+100bp）、P3（-2000～+100bp）、P2（-1000～+100bp）、P1（-500bp～+100bp）和P0（-240～+100bp）等5个调控区片段。预期长度的PCR产物经过测序证实后，定向连接至报告基因载体pGL3-Basic的KpnⅠ和XhoⅠ限制性内酶切位点上，经双酶切鉴定后，成功构建了5个报告基因重组载体pGL3-P0～pGL3-P4。以pSV-β-Galactosidase质粒为内对照，将空载体pGL3-Basic、阳性对照pGL3-Control和不同长度调控区重组质粒分别转染至Glc-82和293A细胞中，培养48小时后检测细胞裂解液中荧光素酶活性。结果显示SPLUNC1基因不同长度调控区片段的荧光素酶活性在Glc-82和293A两种细胞中均存在差异，P0在2种细胞中的转录活性均高于其他长度的片段；在两种细胞中，pGL3-P0的荧光素酶活性分别是空载体pGL3-Basic的5.10和2.69倍，说明P0具有启动子活性，提示SPLUNC1的启动子可能存在于其5’端序列-240～+100bp区域中。此外，SPLUNC1基因的5个调控区片段在Glc-82细胞中的转录活性均高于293A细胞，提示SPLUNC1基因的启动子具有一定的细胞类型特异性。（四）SPLUNC1基因增强子的鉴定以人基因组DNA为模板，采用PCR方法对生物信息学预测的5个增强子片段（E0：-158～-9bp；E1：+3770～+3959bp；E2：+2069～+2201bp；E3：+26454～+6555bp；E4：+14553～+14652bp）进行扩增，PCR产物经过测序证实后，分别定向连接至报告基因载体pGL3-Promoter中SV40启动子上游的KpnⅠ和XhoⅠ限制性酶切位点上和SV40启动子及报告基因下游的BamHⅠ和SalⅠ限制性酶切位点上，经酶切鉴定后，构建了5个报告基因重组载体pGL3-E_0～4-up和5个重组载体pGL3-E_0～4-down。以pSV-β-Galactosidase质粒为内对照，将空载体pGL3-Promoter、阳性对照pGL3-Control和不同增强子重组质粒转染至293T细胞中。测量培养48小时后细胞裂解液中荧光素酶活性的结果显示：当增强子片段位于报告基因启动子上游时，pGL3-E0和pGL3-E2的荧光素酶活性显著增高，调控活性分别是空载体pGL3-Promoter的2.95和3.03倍。当增强子连接于报告基因下游时，pGL3-E0、pGL3-E1和pGL3-E2的荧光素酶活性与对照pGL3-Promoter之间的差异具有显著性，分别是空载体pGL3-Promoter的2.65、2.83和3.13倍。此外，在增强子位于报告基因不同位置的调控活性比较中，片段E1位于报告基因下游时调控活性明显高于其位于报告基因上游时的调控活性。（五）SPLUNC1基因启动子区反式作用因子的鉴定将SPLUNC1基因启动子区分别含有Snail、YY1、n-MYC和HNF-3beta转录因子结合位点的寡核苷酸序列（22～30bp）进行3’端生物素标记，并退火形成双链的探针，与Glc-82细胞的核提取物进行结合，通过凝胶迁移阻滞实验（EMSA）证实Snail、YY1和n-MYC转录因子能与Glc-82细胞核蛋白结合，形成特异性的DNA-protein复合物。此外，染色质免疫沉淀实验（ChIP）中，Snail抗体沉淀的DNA模板，可以特异性地扩增出SPLUNC1基因启动子区片段，进一步证实Snail转录因子参与了SPLUNC1基因的表达调控。结论：1．人SPLUNC1基因的表达具有明显的细胞特异性，它可能是一个与分化有关的基因，而非鼻咽癌的抑癌基因。2．SPLUNC1基因5’端侧翼区-240～+100bp具有启动子活性，并具有一定的细胞特异性。3．SPLUNC1基因的-158～-9bp、+3770～+3959bp和+2061～+2201bp序列具有增强转录的能力。4．Snail、YY1和n-MYC转录因子可能参与了SPLUNC1基因的转录调控。更多还原

【Abstract】 Malignant tumor is a common disease with high incidence, which can severelyendanger mankind’s health. The nature of tumor is a gene disease, and thedevelopment of cancer is a consequence of a series of molecular changes that haveoccurred in cells, which lead to expressing changes of numerous genes. So thepractical therapy for tumor is indeed to be a therapeutic method whose fundamentalpoint is to reverse the genes changed in tumor.Nasopharyngeal carcinoma (NPC) is a malignant tumor with high incidence infive provinces of Southern China and Southeastern Asia. As the other tumor, NPCcarcinogenesis is a multi-cause and multi-step process. To investigate the geneexpression changes in NPC carcinogenesis, He ZW compared the differential geneexpression profiling between human normal nasopharyngeal and NPC tissue by highdensity cDNA microarray and cloned a complete sequence of EST (ExpressedSequence Tags) with relatively special expression in human nasopharyngeal tissue.This new gene was named YH1 gene with the GenBank accession number AF158745.YH1 gene is located at chromosome 20q11.2, covers approximately 7.31kb andconsists of nine exons and eight introns. YH1 cDNA contains a single open reading frame, which encodes a leucine rich (24.6%) protein of 256 amino acids. Becausediffered in splice acceptor sites in their 3’UTRs, there are two transcript variants. YH1is highly homologous to the mouse PLUNC (Palate, Lung and Nasal epitheliumClone) gene, and the size and location of its introns are remarkably conserved withother species, e.g. pig, cow and rat, so YH1 has been cataloged to PLUNC family.Due to its size is the smallest one in the PLUNC family, YH1 has been nowdesignated SPLUNC1 (short PLUNC 1).SPLUNC1 gene had the relative tissue expression specificity. SPLUNC1 usuallyis expressed in human nasopharyngeal tissues, but very low or rare in NPC tissues. Itis also expressed in human trachea but a little bit in right atrium, jejunum, lung,salivary gland and fetal lung tissue and rarely in others tissues. Furthermore, LUNX, atranscript variant of SPLUNC1, is highly expressed in non-small cell lung cancer(NSCLC), and was detected in lymph nodes diagnosed as NSCLC early-stagemetastasis by RT-PCR, so LUNX mRNA is a possible molecular marker ofmicrometastasis in NSCLCs.The function of SPLUNC1 is unknown. The results from bioinformatics analysisshows that the SPLUNC1 protein contains a signal peptide at the N terminus, and theamino acid sequence is most closely similar to the secretary proteins produced by thesalivary glands and glandular epithelium of the trachea, the von Ebner minor salivaryprotein (also known as LCN1) and the parotid secretary protein. A bactericidal/permeability-increasing protein (BPI) domain was predicted in the three-dimensionalstructure of SPLUNC1 protein. The full-length SPLUNC 1 protein was found to bestructurally related to BPI which is the bactericidal protein and capable ofneutralizing the endotoxin. Based on the structure and functional domain ofSPLUNC1 protein, it was predicated to have the bactericidal and binding function oflipopolysaccharide. Furthermore, SPLUNC1 was identified to be a genetic risk factor for NPC in the study that single nucleotide polymorphisms (SNPs) in SPLUNC1 weresystematically screened and found to be associated with susceptibility to NPC in theChinese population.The regulatory mechanism of gene expression is an important field of researchin post-genomic era. The exact gene expression depends on very complicatedregulatory mechanisms which mainly happen at transcriptional level. Promoter, aspecific DNA sequence capable of binding with the RNA polymerase, is a necessarycis-acting element for the gene transcription. Gene expression is often determined bythe initiation process of specific promoter in the regulation of gene expression.Enhancer, also a positive cis-acting element for the gene transcription, plays apromotion role for the promoter to regulate its downstream gene, and assure the exactexpression of the downstream gene accompanying with other negative cis-andtrans-acting factors (transcription factors). Different transcription factors respond todifferent stimulation by the environment outside or different signals in differentdevelopment stages, and then combine with the transcriptional acting elements, whichactivate or inhibit the gene expression, hence the gene expression is regulated. Theexact expression of a gene relies on the cooperation of cis-acting element,trans-acting factor and RNA polymerase. Nowadays, on the basis of illuminating themechanisms of transcriptional acting, it has developed into a new way for thetreatment of tumors that by selectively interfering the combination of sometranscriptional factors with DNA, abnormal gene expression is reversed, and thenanti-cancer effect is realized.Based on the advanced research ofSPLUNC1 gene and its potential significancein the mechanism of tumorigenesis and prevention & treatment of tumor, identifyingthe exact cell types that expressed SPLUNC1 and the promoter sequences andtranscription factors essential for transcriptional regulation of SPLUNC1 gene may greatly contribute to understanding the regulatory mechanism of its expression.Furthermore, a tissue/cell-specific promoter constructed basing on the specificexpression of SPLUNC1 will offer a new way for prevention and control of tumor.We made some researches to clarify the characteristics and regulatory mechanism ofSPLUNC1 gene expression as follows:1. Identification of cell types expressed SPLUNC1 geneIn situ hybridization method was used to detect the expression of SPLUNC1gene in nearly 30 kinds of tissues. The results showed that SPLUNC1 gene wasexpressed neither in squamous epithelial cells of palate, epidermis, esophagus, andthe junction of esophagus with cardia of stomach, nor in the tumor cells ofnasopharyngeal poorly-differentiated squamous cell carcinoma, esophageal squamouscell carcinoma, and lung squamous cell carcinoma, nor in the metaplastic squamouscells in nasopharynx, trachea and uterus cervix. SPLUNC1 gene also was notexpressed in the single layer columnar epithelia cells in stomach, gallbladder, jejunum,colon, endometrium and cervix. It was expressed mainly in pseudostratified columnarepithelial cells in nasopharynx, trachea and bronchi, and interestingly, its expressionwas gradually down-regulated in respiratory tract from upper to lower end, and noexpression was found in lung tissues in the end. SPLUNC1 gene was also found notonly in duct and serous gland cells both in parotid and submandibular gland, but alsoin cells of submucosal serous gland in nasopharynx, lung and esophagus, while noexpression were found in cells of mucosal gland. The expression of SPLUNC1 wasdetected in parietal cells of gastric submucosa and epithelial cells of lobula and ductof mammary gland. In addition, SPLUNC1 gene was found strongly expressed inwell-differentiated adenocarcinoma cells in lung, stomach, colon, mammary gland,uterus endometrium and cervix. All these data suggests that SPLUNC1 is a geneprobably associated with cell differentiation, not a tumor suppressor gene in nasopharyngeal carcinoma, and its expression is highly cell specific. These results laya favorable basis for further studying the regulatory mechanisms of its expression andbiological function, and shed light on the diagnosis and treatment of tumor.2. Prediction of the regulatory elements of SPLUNC1 gene by bioinformatics toolsBy analyzing the 6kb sequence from -5000bp to 1000bp (the first nucleotide of thepublished cDNA is +1bp) of SPLUNC1 by BLAST alignment, promoter predictionsoftware and recognition of transcription factor binding sites, the results showed thetranscription start site is located at the region of -1～+10bp, a TATA box presents atthe -29bp position, and the promoter exists in the region from -490 to +89bp ofSPLUNC1. The conserved region was found in the promoter sequence betweenhuman and mouse species, especially from -245 to -1bp, with some transcriptionfactor binding sites. In addition, Enhancer element location (EEL) software predictedthat five highest scoring cis-module resulting from the alignment of 30kb human andmouse sequences was located to 5’ end, interior and 3’ end of SPLUNC1. Predictedresults are perhaps favorable o identify all possible regulatory regions.3. Regulatory activity assay for the 5’ flanking sequence of SPLUNC1The expression of SPLUNC1 mRNA was detected by Real-time reversetranscription polymerase chain reaction (RT-PCR) in eleven cell lines includinghuman lung adenocarcinoma-derived cell line (Glc-82), NPC-derived cell lines (5-8F,6-10B, C666-1, CNE-1, CNE-2, HNE-1 and HONE-1), human embryonic kidney cellline (HEK293A), et al. The expression level of SPLUNC1 was the highest in Glc-82cells, which provided a suitable cell model to study the regulatory mechanisms ofSPLUNC1.Based on genomic sequence covering 5’end of SPLUNC1 gene, we designedfive pairs of primers that the downstream end was the first exon(+1) down stream 100bp and the end of upstream located at -3000, -2000, -1000, -500, and -240bp,respectively. The five fragments, P4 (3100bp), P3 (2100bp), P2 (1100bp), P1 (600bp)and P0 (340bp) were isolated from the human genomic DNA by PCR amplification.After the expected PCR products were cloned into T vector and confirmed by directsequencing and enzymatic digestion, the sticky-end fragments digested by KpnⅠ/XhoⅠwere inserted into PGL3-Basic, a promoter-less luciferase reporter vector to form fiveplasmid constructs, pGL3-P0～pGL3-P4. Transient transfections included reporterplasmids, pGL3-Basic, pGL3-Control as positive control and pSV-β-Galactosidasereporter vector as an internal control. The promoter activity of each construct wasevaluated by luciferase assay of cell extracts from Glc-82 and 293A transfected 48hours later. We determined that the differences among the transcriptive activity of fivepromoter fragments in Glc-82 and 293A cells, and found that the transcriptiveactivities of P0 in both cell lines were higher than that of other fragments. Thetransfected reporter genes pGL3-P0 displayed an enhanced expression of luciferaseactivity by 5.10-fold and 2.69-fold compared with the blank vector pGL3-Basic inGlc-82 and 293A cells, respectively. These results indicated that the 5’ flankingregion from -240 to +100bp manifested relatively low but functional promoteractivity, and the promoter of SPLUNC1 gene might be located at this region.Furthermore, taking the luciferase activity of pGL3-Control as 100%, the luciferaseactivity of the five fragments in Glc-82 cells were higher than that in 293A,suggesting the promoter of SPLUNC1 is tissue specific.4. Identification of SPLUNC1 enhancersBased on the human genomic sequence, five pairs of primers targetingenhancers’ fragments (E0-E4) predicted by bioinformatics were designed and thenisolated from the human genomic DNA by PCR amplification. After the expectedPCR products were confirmed by direct sequencing, the sticky-end fragments, digested by KpnⅠ/XhoⅠand by BamHⅠ/SalⅠrespectively, were inserted intoPGL3-Promoter, a reporter vector, to form five reporter plasmid constructs,pGL3P-E0～4-up, and five plasmid constructs, pGL3P-EO～4-down. Transienttransfections to 293T cells included all reporter plasmids, pGL3-Promoter,pGL3-Control as positive control and pSV-β-Galactosidase reporter vector as aninternal control. The enhancer activity of each construct was evaluated by luciferaseassay of cell extracts transfected 48 hours lated. The results showed that when theenhancer fragment was located at the upstream of reporter gene promoter, theregulatory activity of enhancer E0 and E2 were highly increased, which was 2.92-foldand 3.03-fold higher compared with that of pGL3-Promoter, respectively, whileenhancer fragments were located at the downstream of reporter gene, the regulatoryactivity of enhance E0, E1, and E2 were highly increased, which was significantlyhigher than that of pGL3-Promoter (the difference was 2.65-fold, 2.83-fold, 3.13-foldrespectively). In addition, in comparison of regulatory activity of enhancer fragmentsin different reporter gene locations, the activity of fragment E1 located at thedownstream of reporter gene was obviously higher than that at the upstream ofreporter gene.5. Identification of trans-acting factors in the promoter of SPLUNC1The 3’ end of 22～30bp oligonucleotide DNA sequences derived from thepromoter of SPLUNC1 containing the consensus motifs of Snail/YY1/n-MYC/HNF-3beta were labeled with biotin. The biotin-labeled complementaryoligos were annealed to form double-stranded probe for elcetrophoretic mobility shiftassays. The nucleic protein was extracted from Glc-82 cells and bound to the labeledprobe with unlabeled specific-competitive and unspecific-competitiveoligonucleotides. A specific band was observed in the gel with probes containing aputative Snail site and unaffected by excessive mutant probe, while no specific band was observed with excessive non-labeled probe. Similar band patterns were observedusing probes containing the binding sites of YY1 and n-MYC, while no specificbinding was found in the HNF-3beta reaction. These results suggest that Snail, YY1and n-MYC proteins bind to the binding sites located at the proximal region ofSPLUNC1 promoter. To further confirm that Snail could bind to the SPLUNC1promoter, chromatin immunoprecipitation assays (CHIP) was carried out. AfterGlc-82 cells were cross-linked with formaldehyde and incubated with Snail antibody,DNA-protein-antibody complexes were precipitated. DNAs interacted with Snailprotein were purified and were determined to have the -240 to +100bp and -157 to+100bp region of human SPLUNC1 promoter.Conclusions:1. SPLUNC1 is a gene probably associated with cell differentiation, not a tumorsuppressor gene in nasopharyngeal carcinoma, and its expression is highly cellspecific.2. The 5’ flanking region from -240 to +100bp of SPLUNC1 gene manifestedfunctional promoter activity and cell specificity.3. The DNA sequences from -158 to -9bp, +3770 to +3959b and +2061 to +2201bphad the activity promoted transcription.4. The transcription factors, Snail, YY1 and n-MYC, maybe participate in theregulation process of transcription of SPLUNC1 gene.更多还原