【作者】 王彬；

【导师】 颜其贵；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2016， 博士

【摘要】 羊地方性鼻内腺瘤(Enzootic Nasal Adenocarcinoma,ENA)又称羊地方性鼻内肿瘤(enzootic nasal Tumor,ENT)或山羊传染性鼻内腺癌,是由地方性鼻内肿瘤病毒(Enzootic Nasal Tumor Virus of Goats,ENTV-2,Enzootic Nasal Tumor Virus of Sheep, ENTV-1)引起的一种慢性、进行性、接触性传染病,除澳大利亚和新西兰外,该病呈世界性分布。临床表现为食欲减退,极度消瘦,呼吸困难,鼻漏,出现鼻内单侧或双侧性增生物。该病发病率为5%-15%不等,一旦出现临床症状,几乎都以死亡告终。迄今为止该病没有有效的方法进行早期诊断,一旦出现临床症状就只能淘汰,也不能区分潜伏感染的羊只,导致疾病在整个羊群中散布,严重地威胁羊群的安全。至今,还未能成功建立ENTV的体外培养体系,使得研究该病毒的致瘤机制和免疫特性遇到了阻碍。本研究对ENTV-2及其对应的内源性逆转录病毒进行了全基因组测序,并进行了比对分析,分析了ENTV-2在病毒基因层面的致瘤机制及鉴别内外源ENTV-2的靶基因位点；通过对ENA肿瘤组织及癌旁组织进行高通量测序,建立了miRNA文库,分析miRNA在山羊鼻肿瘤组织和癌旁组织中的表达差异,利用生物信息学软件分析差异性表达miRNA的靶标及相应的生物学功能,分析miRNA在肿瘤形成中的作用,为进一步研究miRNA在ENA中的作用和机制提供理论和实验依据,在表观遗传层面研究ENTV-2的致瘤机制。1.山羊致病性鼻内腺瘤病毒全基因组序列测定及分析通过分段RT-PCR方法从6例ENA山羊鼻内肿瘤组织中扩增出6条ENTV-2全基因序列。ENTV-2 CHN6全长7500nt, ENTV-2 CHN7全长7501nt,ENTV-2 CHN8全长7501nt,ENTV-2 CHN9全长7503nt,ENTV-2 CHN10全长7500nt,ENTV-2 CHN11全长7499nt。将所得序列与GenBank中收录的ENTV-2和ENTV-SC进行全基因比对,6个全基因组序列与ENTV-2的同源性均高于87%,与ENTV-SC的同源性均高于91%,具有较高的同源性,并且具有ENTV的gag和env基因的特征区域。经过比对,发现ENTV-2和ENTV-1的基因组的突变区域主要集中在LTR的U5、U3区域,gag和env区域。ENTV-2与JSRV的基因组同源性约为84-89%,ENTV-1与比对的JSRV的基因组同源性为88.4-89.6%。ENTV-1与ENTV-2和JSRV的同源性比较接近。突变区主要集中在170bp以前和7400bp以后,即突变区主要集中在U5区、U3区和env基因末端。6株病毒株ENTV-2 CHN6-ENTV-2 CHN11的核苷酸突变以点突变为主,只有LTR区有2bp的插入突变和2bp的缺失突变,编码区的突变均为点突变,但突变并未引起编码蛋白的结构域和功能的改变。分析发现,6株病毒株ENTV-2 CHN6-ENTV-2 CHN11的基因组存在点突变导致的变异,经生物软件分析突变尚未积累到影响病毒的结构蛋白的空间构型的改变。2.山羊内源性逆转录病毒全基因组序列测定及分析通过分段PCR方法,从6例ENA山羊肾脏中扩增出6条enENTV。enENTV-CHN 1全长7889bp,enENTV-CHN2全长7890bp,enENTV-CHN3全长7897bp,enENTV-CHN4全长7891bp,enENTV-CHN5全长7928bp,enENTV-CHN6全长7921bp。将所得序列与GenBank中收录的ENTV-2和ENTV-SC进行全基因比对,6个全基因组序列与ENTV-2的同源性均高于87%,与ENTV-SC的同源性均高于91%,具有较高的同源性,并且具有ENTV的gag和env基因的特征区域。enENTV与同源ENTV的核苷酸突变区主要集中在U3、U5、gag 3’端和env 3’端。通过全基因组同源性分析及序列分析发现enENTV遗传比较稳定,本次获得enENTV-CHN1～6突变具有地域差异但与性别相关性不明显。通过全基因组序列比对发现enENTV与ENTV-2相比具有5处连续突变,该区域可以作为区别鉴定enENTV与ENTV-2靶基因位点。3. ENTV-2 RT-PCR检测方法的建立通过全基因组序列比对发现enENTV与ENTV-2相比在LTR U3区具有5处连续突变,7453bp处ENTV-2 CHN6-11有7bp插入突变,7828bp处3bp插入突变,针对ENTV-2相对enENTV的插入突变区设计了一对引物,对鉴别ENTV-2与enENTV的靶基因位点进行了验证,建立ENTV的快速RT-PCR检测方法。用该引物进行RT-PCR扩增,结果得到与试验预计相符的391bp的特异性扩增条带,与ENTV-2CHN6-11比对同源性均高于99%；此方法对健康羊鼻粘膜上皮、肺炎链球菌和支原体的扩增结果均为阴性,最低能检测10 pg的RNA,有较好的稳定性和重复性；对保存的100份病料进行检测,并与临床诊断结果进行比较,符合率100%。试验表明全基因组序列比对发现的靶基因位点可以用于ENTV-2与enENTV的鉴别。4.山羊鼻内腺瘤miRNA测序及表达差异分析应用Illumina高通量测序技术,对ENA山羊鼻内腺瘤及癌旁组织进行miRNA测序,建立miRNA文库,并对山羊鼻内腺瘤组织和癌旁组织中miRNA的表达差异进行了分析。利用生物信息学软件分析差异性表达miRNA的靶标及相应的生物学功能,进一步分析miRNA在肿瘤形成中的作用。本试验发现406条已知miRNA,29条新miRNA,其中只在肿瘤组或对照组表达的有34条(包含3条新miRNA),在肿瘤组和对照组中有116条miRNA有表达差异显著,与对照组比较,其中54条miRNA在肿瘤组中表达下调,60条miRNA在肿瘤组织中表达上调,有2条只在对照组中表达。另外,在功能分析中,预测出显著差异表达121 miRNA(116条显著差异表达的miRNA和5条star序列)的靶标、6172条、1792条显著富集GO和97条显著富集KEGG pathway、GO和KEGG pathway功能分析显示大部分基因涉及到细胞分化、信号传导等重要生命过程。本试验第一次对山羊ENA中niRNA进大规模鉴定,为阐明复杂而精确的miRNA-mRNA调控网络和ENA的发展过程的研究提供参考。利用荧光定量PCR技术对随机挑选的9个miRNA进行验证,结果表明9个：miRNA的表达趋势与测序结果一致,说明本次构建的miRNA文库获得的测序数据可靠。chi-mir-133a、chi-mir-145-5p、mchi-mir-146a/200a可能作用于靶基因BRAF而通过对MAPK信号通路、PI3K-Akt信号通路、Ras信号通路,影响细胞的正常分裂周期,促进了细胞的增殖,对ENA的发生起到了促进作用。chi-mir-874-3p作用于靶标VEGFA、chi-mir-148a-3p作用于靶标TGF-β-RAP1,通过Pathways in cancer使得肿瘤细胞逃脱了TGF-β的抑制生长作用,对ENA的发生、发展创造了条件,低表达的金属蛋白酶和TGF-β在ENA的浸润转移中发挥了抑制作用。更多还原

【Abstract】 Enzootic nasal adenocarcinoma (ENA) as an epithelial tumor, caused by enzootic nasal tumor virus (ENTV:Enzootic Nasal Tumor Virus of Goats. ENTV-2; Enzootic Nasal Tumor Virus of Sheep, ENTV-1) is a chronic, progressive, contact sexually transmitted diseases. The clinical symptoms are loss of appetite, extremely thin, dyspnea, rhinorrhea, nasal unilateral or bilateral puffiness. The disease incidence of 0.1% to 15%, once the clinical symptoms appearance, almost all ended in death. Except Australia and New Zealand. this disease has been spread throughout almost all major countries to infected goats. So far no methods are effective for early diagnosis and the goat can only be eliminated after symptoms appear. More seriously, because it cannot distinguish between latent infections and health, disease spread in the group, infecting a greater number of goats, even threaten the security of population. It never rains but it pours, to establish a system of cultivation ENTV in vitro has failed yet, which is an obstacle to reveal tumorigenic mechanism as well as the virus immunological characteristic.In this research. ENTV-2 and its corresponding endogenous retroviruses were whole-genome sequenced and comparative analysis carried out to analyze the ENTV-2 gene in the virus level in tumorigenesis and target gene for identifying of internal and external sources ENTV-2:Hiseq sequencing ENA tumor tissues and adjacent tissues was used to establish the miRNA library, analysis differentially expressed miRNA between cancer tissues and tumor-adjacent tissues and use bioinformatics software analysis differentially expressed target and the corresponding biological function of miRNA, analyze the role of miRNA in tumor formation, and provide theoretical and experimental basis for further study of the role and mechanism of miRNA in the ENA, the epigenetic inheritance level tumorigenic mechanism of ENTV-2.1. Sequence Analysis ENTV GenomeIn order to amplify ENTV s full genome. RT-PCR were used to amplify fragment from 6 ENA goat nasal tumor tissue. ENTV-2 CHN6 full length 7500nt, ENTV-2 CHN7 full length 7501nt, ENTV-2 CHN8 full length 7501nt, ENTV-2 CHN9 full length 7503nt, ENTV-2 CHN10 full length 7500nt, ENTV-2 CHN11 full length 7499nt. The resulting sequence in GenBank ENTV-2 and ENTV-SC-wide gene comparison, six complete genome sequence homology ENTV-2 was higher than 87%, and ENTV-SC was high homology at 91%, with higher homology, and has a feature region ENTV the gag and env genes. After alignment ENTV-2 and ENTV-1, mutations in regions of the genome focused on the LTR U5, U3 region, gag and env regions. ENTV-2 genome and JSRV homology of about 84-89%, ENTV-1 ratio for the JSRV genome homology of 88.4-89.6%. ENTV-1 with ENTV-2 and JSRV homology closer. Mainly concentrated in the region of the mutation mainly in the U5 region of the mutation region, and the end of the env gene U3 region.The main nucleotide mutation of ENTV-2 CHN6-11 is point mutation, only LTR region has insertion mutation 2bp and 2bp deletion mutations, all the mutations are point mutations in the coding region, but did not cause mutations encoding protein domains and functional change. It can be considered ENTV-2 CHN6-11 genomic variation caused by the presence of point mutations, but mutations have not yet accumulated to change the structure of the protein changes and function of the virus.2. Sequence analysis goat endogenous retrovirus genomeIn order to amplify enENTV s full genome, PCR was used to amplify fragment from 6 ENA goat kidney. enENTV-CHN1 full length 7889bp, enENTV-CHN2 full length 7890bp, enENTV-CHN3 full length 7897bp, enENTV-CHN4 full length 7891bp, enENTV-CHN5 full length 7928bp, enENTV-CHN6 full length 792 lbp. The resulting sequence in GenBank ENTV-2 and ENTV-SC-wide gene comparison, six complete genome sequence homology ENTV-2 was higher than 87%, and ENTV-SC was high homology at 91%, with higher homology, and has a feature region ENTV the gag and env genes. Mutation regions between enENTV and ENTV are mainly in U3, U5, gag 3’end and env 3’end. Through whole-genome sequence homology analysis and genetic analysis enENTV s genetic is relatively stable. The mutation of enENTV-CHN1～6 has regional differences, degree of sex difference is not obviously. Through whole-genome sequence alignment we find enENTV compared with ENTV-2 has five consecutive mutations, which can be identified as the difference between enENTV with ENTV-2 target gene locus.3. Establishment of RT-PCR detection method for ENTV-2Through whole-genome sequence alignment of enENTV and ENTV-2 discovery five consecutive mutations in LTR U3 region, wherein at 7436bp ENTV-2 CHN6-11 have 5bp insertion mutation, at 7453bp ENTV-2CHN6-11 have 7bp insertion mutation, at 7828bp 3bp insertion mutation. A pair of primers was designed for differential ENTV-2 of the target gene locus enENTV at insertion mutation region. A rapid RT-PCR detection of ENTV was verified. The primers used RT-PCR amplification, and the trial is expected to match the results of specific amplification bands of 391 bp. compared with ENTV-2 CHN6-11 homology were higher than 99%; this method to nasal healthy sheep epithelium. Streptococcus pneumoniae and mycoplasma amplification results were negative, the minimum can detect 10 pg RNA, better stability and repeatability:the preservation of 100 clinical samples for testing, and with the clinical diagnosis comparison, was 100%. Test showed that whole-genome sequence alignment target gene identified may be used to identify the ENTV-2 and enENTV.4. Analysis of Differentially Expressed MicroRNA in ENA of GoatThis study was based on the differences between ENA tumor and para-carcinomas nasal tissues, through small RNA Illumina high-sequencing and RT-PCR. to dig miRNA of goat nose organization and to illustrate its differential expression, furthermore combined with the target gene prediction, function analysis, to prove the role of miRNA in tumorigenesis. As a result,406 known miRNAs and 29 novel miRNAs including 34 miRNAs (3 novel miRNAs) only expressed in control group or cancer group.116 miRNAs were significant difference in control group and cancer group with 54 decreased.60 increased and 2 miRNAs expressed in control group. About function analysis, including 6172 non-redundancy target genes.1792 significant GO and 97 significant KEGG pathway for 121 miRNAs (including 116 significant expression miRNAs and 5 star sequence)were predicted. GO and KEGG pathway analysis showed that the majority of targets were involved in cell proliferation, signal transduction. etc. important life activities. This research did the first large-scale identification of miRNAs in the ENA to enrich the repertoire about Capra hircus and to lay a theoretical basis for stating the complicated and precise miRNA-mediated regulatory networks for gene expression in the pathology and develop progression of the enzootic nasal adenocarcinoma.chi-mir-133a, chi-mir-145-5p, mchi-mir-146a/200a may act on target genes by BRAF and MAPK signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, affecting the normal cells division cycle, promoting cell proliferation, the formation of ENA has played a catalytic role. chi-mir-874-3p act on target VEGFA, chi-mir-148a-3p acting on target TGF-β-RAP1, probably by Pathways in cancer makes the tumor cells to escape growth of TGF-β inhibition of ENA occurrence and development to create the conditions low expression of metalloproteinases and TGF-β inhibition play in the invasion and metastasis of ENA.更多还原