| 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-?-RAP1, probably by Pathways in cancer makes the tumor cells to escape growth of TGF-? inhibition of ENA occurrence and development to create the conditions low expression of metalloproteinases and TGF-? inhibition play in the invasion and metastasis of ENA. |
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