Identification MicroRNAs In Brain Of Cattle And Buffalo By High Throughput Sequencing

microRNAs(miRNAs)are a class of endogenous small RNA molecules approximately 19?23 nt in length.miRNAs can cause degradation or translational inhibition by binding to the 3' untranslated region(3'UTR)of target genes,so they regulate expressional levels of genes at post-transcriptional level.miRNAs are widely present in a variety of organisms and are high conservation among species.miRNAs play important roles in a variety of key biological processes,including cell development,differentiation,proliferation,aging,apoptosis,signal transduction,metabolic processes and diseases.Previous studies have shown that miRNAs are closely involved in brain function loss,and the development and progression of neurodegenerative diseases,such as Alzheimer's disease,Parkinson's disease,Huntington's disease and prion diseases.Bovine spongiform encephalopathy(BSE)is a compelling prion disease,it can cause functional damage and physiological lesions in bovine brains.Until now BSE cases have been reported worldwide in nearly 0.2 million Bos taurus cattle,but never in the domestic buffalo,Bubalus bubalis.Our previous studies have shown that there are significant differences in both genetic background and expression of genes associated with BSE between cattle and buffalo.Importantly,we found prion protein(PrP)that plays a crucial role in BSE had expressional differences between the two species.This phenomenon may be due to miR-125b-5p,miR-132-3p,mi-145-5p,miR-331-3p and miR-338-3p specifically regulate the buffalo PRNP gene 3'UTR.Whether there are different expression levels of miRNAs in brain tissues between cattle and buffalo?And whether these miRNAs directly regulate the expression of some key genes,and potentially involve in the pathways of brain function and neurodegenerative diseases?However,there remains much unknown about these aspects.Therefore,the aim of study was to identify miRNAs and to analyze the expressional differences of miRNAs between five cattle and five buffalo brain tissues by Illumina HiSeq2000 high-throughput sequencing technology,and to identify and screen miRNAs that may bind to the genes associated with brain function and neurodegenerative diseases.Furthermore,the expression levels of the observed miRNAs were validated by stem-loop RT-qPCR method in large samples.The regulations of predicted target genes by these miRNAs were also identified by luciferase reporter assays.1.Sequencing results showed that 11,724,317 and 11,872,170 clean reads were obtained sampled from cattle and buffalo brain tissues respectively,accounting for 99.37%and 99.36%of the total high quality reads.A total of 451 known miRNAs and 118 novel miRNAs were found.Among of these miRNAs,there were 59 known miRNAs and 61 novel miRNAs expressional differences between cattle and buffalo brains,respectively.2.The prediction of target gene revealed that most of miRNAs have several target genes,and these target genes are widely expressed in animals' brains.GO analysis showed that these target genes are involved in a variety of physiological processes,including biological transcription,apoptosis,and binding.KEGG analysis showed that all target genes can be mapped to more than 300 pathways.We focused on four pathways related to neurodegenerative diseases,because many target genes were enriched in the pathways.The pathways are associated with prion disease,Alzheimer's disease,Huntington chorea and Parkinson's disease.3.After predicting miRNA that may bind to the 3'UTR of reported gene,we selected five known miRNAs(bta-miR-129-3p,bta-miR-92a,bta-miR-935,bta-miR-877 and bta-miR-874)and seven novel miRNAs(novel-miR-26,novel-miR-13,novel-miR-5,novel-miR-25,novel-miR-40,novel-miR-35 and novel-miR-4)to perform expression analysis by stem-loop RT-qPCR method.The result showed that the RT-qPCR results of 12 miRNAs expression were similar to the sequencing results.4.Furthermore,luciferase reporter assays showed that some genes(CASP3,PSENEN,NEURL4,BRI3,CDK5R1,ZBTB24 and ZNF292)relative to brain function,neuronal differentiation and development,as well as neurodegenerative diseases can be directly regulated by five miRNAs(bta-miR-935,bta-miR-877,novel-miR-13,novel-miR-25 and novel-miR-4).For example,novel-miR-25 can regulate ZBTB24 expression,which was reported to involve in the formation of dendrites in brain neurons,and to affect brain function and brain disease;novel-miR-13 can regulate the expression of BRI3 and CDK5R1 genes,which were found to affect the differentiation and development of neurons,and to be associated with Alzheimer's disease.The present study is systematic identification of miRNA of cattle and buffalo brain tissues.These results not only enriched the information of bovine miRNAs,but also obtained many differently expressed known and novel miRNAs between cattle and buffalo brains.The known miRNAs and novel miRNAs could regulate the expression of the important genes associated with brain neuron functions at post-transcriptional levels.The findings of this study may provide a new idea to study the prevention and treatment of BSE.