【摘要】 目的:基于高通量测序结合生物信息学挖掘人新miRNA分子,以期为疾病诊治提供理论基础。方法:分别提取4例正常人和乳腺癌患者的血清外泌体,通过Illumina HiSeq SE50高通量miRNA测序,滤掉已知miRNA后,用Bowtie 2筛选出与人全基因组数据库(GRCh38.P13)匹配的序列,再用MIREAP和RNAfold软件筛选出新miRNA候选序列。分别收集18例正常人和29例乳腺癌患者血清,用实时荧光定量PCR(qRT-PCR)分析其中两个新miRNA的表达水平。最后通过miRWalk 2.0数据库对一个miRNA分子进行靶基因预测,并进一步利用GEPIA和UALCAN数据库预测靶基因在乳腺癌中的临床意义。结果:提取得到的外泌体质量良好。通过筛选得到7条新miRNA的候选序列,其中miRNA-A和miRNA-B在正常人和乳腺癌患者血清中均有表达,但无显著差异。预测得到的miRNA-A靶基因包括CLK1、CLK4、GNG2和NPR2,它们在乳腺癌组织中均低表达,并且其表达水平在乳腺癌的不同分子分型和组织类型存在显著差异。结论:经外泌体高通量miRNA测序筛选并已初步验证得到两个新miRNA分子,其功能有待进一步研究。更多还原

【Abstract】 Objective: To discover new human miRNA molecules based on high-throughput sequencing combined with bioinformatics, providing a theoretical basis for disease diagnosis and treatment. Methods: The serum exosomes of 4 normal individuals and breast cancer patients were extracted. The miRNA profile of serum exosomes was analyzed by Illumina HiSeq SE50 high-throughput sequencing. After filtering out known miRNAs,Bowtie 2 was used to screen out the sequences that matched well in the human genome database(GRCh38.P13),and then MIREAP and RNAfold software were used to screen out new miRNA candidate sequences. The serum from 18 normal individuals and 29 breast cancer patients were collected, and the expression levels of two new miRNA molecules were analyzed by real-time fluorescent quantitative PCR(RT-qPCR). Finally, the miRWalk 2.0 database was used to predict the target genes of a new miRNA molecule, and the GEPIA and UALCAN databases were used to predict the clinical significance of target genes in breast cancer. Results: High-quality exosomes can be obtained from human serum. Seven new miRNA sequences were obtained after screening. miRNA-A and miRNA-B were expressed in healthy people and breast cancer patients, but there was no significant difference between them. The predicted miRNA-A target genes included CLK1, CLK4, GNG2 and NPR2. The genes were down-regulated in breast cancer tissues. Moreover, the expression levels of them were significantly different among breast cancer patients with different molecular subtypes. Conclusion: Two new miRNA molecules have been preliminarily verified by exosomal high-throughput miRNA sequencing, and their functions need to be further explored.更多还原