Analysis Of Differentially Expressed MicroRNA In Enzootic Nasal Adenocarcinoma Of Goat

Enzootic nasal adenocarcinoma (ENA) as an epithelial tumor, caused by enzootic nasal tumor virus(ENTV)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%, So far no methods are effective for early diagnosis and once the clinical symptoms appearance, almost all ended in death. More seriously, 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.MicroRNA(miRNA) is a class of noncoding RNA about 21-24 nt. It involved in various types of cancer, including human nasopharyngeal carcinoma, and plays an important physiological role. At present, it is a hotspot in cancer research.Our research is based on the close relationship between tumor and miRNA. Through the illumina high-throughput sequencing and qPCR technology, using of bioinformatics software and information in NCBI database to excavation functions and signal pathways of targets of differentially expressed miRNA in tumor groups.The effective sequences were blasted with Rfam and EST database by bioinformatics software to remove non-miRNA,the remaining sequences were blasted with goat genomic to get the new miRNA.The RNAfold software was used to identify the mature structure and precursor of the new miRNAs and using miRDeep software evaluates the prediction results according the false positive rate.TPM metrics calculate the expression of miRNA in tumor group and control group.The DESeq software was used to screen the differentially expressed miRNA according to p<0.05 and greater than or equal to 2 folds. Using miranda algorithm to assess scores and energy stability from miRNA-3’UTR sequence,the threshold parameters are Scores>150,AG<-30 kcal/moland demand strict 5’seed pairing,the target genes of differentially expressed miRNA were predicted.Combined with the NCBI database information, using Gene ontology, KEGG pathway analysis software, to get targets functional annotation and classification, also the related signaling pathway,which help to reveal the role of miRNA in tumor formation. This study was based on the differences between goat 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. Designing the primers according to the sequence provide by illumina high-throughput sequencing, we verify the 7 known miRNA and 2 new miRNA by applicating of fluorescence quantitative PCR. As a result,406 known miRNAs and 29 novel miRNAs including 34 miRNAs (3 novel miRNAs) expressed in control group or cancer group have been found.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 6925 non-redundancy target genes,1792 significant GO and 97 sinificant KEGG pathway for 121 miRNAs (including 116 significant expression miRNAs and 5 star sequence)were predicted.GO and KEGG pathway analysis showed that according to the percentage, the top six signaling pathways are MAPK signaling pathway、Ras signaling pathway、prolactin signaling pathway、Pathways in cancer、Viral carcinogenesis and MicroRNA in cancer, the majority of targets were involved in cell proliferation and apoptasis,metabolism, signal transduction, etc.important life activities. We predict the effect of chi-mir-874-3p on the target VEGFA and the effect of chi-mir-148a-3p on the target TGFβRAP1, may creating conditions for the tumor cells escape the inhibition of TGFβAbout the invasion and metastasis of ENA, the low expression of metalloproteinases and Syndecan may play a suppressing role.qRT-PCR confirme the expression of the nine miRNAs between ENA and the para-cancerous tissues with an increased sample size. The expression trend of eight miRNAs is in accord with Illumina High-Throughput Sequencing, one miRNA(chi-miR-218) both in sequencing and qPCR verification have shown a down-expression in tumor group, but the down-expression multiple is different.Two new miRNA amplified successfully,which imply the sequencing quality is reliable. 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.