非小细胞肺癌患者外周血外泌体中差异表达的微小RNAs研究

Differentially Expressed MicroRNAs in Peripheral Blood Exosomes in Patients with Non-small Cell Lung Cancer

背景目前,临床上尚缺乏早期、有效诊断非小细胞肺癌(NSCLC)的指标,来源于癌组织的外泌体及其内部物质因可反映亲本细胞性质而有望成为早期诊断肿瘤的生物学标志物。目的通过高通量测序技术筛选NSCLC患者外周血外泌体中差异表达的微小RNAs(miRNAs)。方法收集2018年1月-2019年1月于深圳市人民医院就诊的4例NSCLC患者的外周血样品,其中肺腺癌(LUAD)2例、肺鳞状细胞癌(LUSC)2例;另收集同期4例健康志愿者的外周血样品作为对照。采用HiSeq 2500高通量测序仪进行miRNAs测序,采用DESeq2软件筛选差异表达的miRNAs;在癌症基因组图谱(TCCGA)中选取并比较39对LUAD组织及其配对癌旁正常组织、45对LUSC组织及其配对癌旁正常组织miRNA-9-3p测序结果。结果(1)高通量测序结果显示,4例NSCLC患者外周血外泌体中平均检测到776种miRNAs,4例健康志愿者外周血外泌体中平均检测到238种miRNAs。(2)LUAD与LUSC患者外周血外泌体中差异表达最显著的10种miRNAs中有6种是重合的,分别为miRNA-1228-5p、miRNA-129-5p、miRNA-760、miRNA-885-3p、miRNA-9-3p、miRNA-95-3p。(3)LUSC-1、LUSC-2、LUAD-1及LUAD-2外周血外泌体中miRNA-9-3p表达量分别为71.51、15.61、13.99、16.11转录本数,4例健康志愿者外周血外泌体中miRNA-9-3p表达量均为0。TCCGA中LUAD组织和LUSC组织miRNA-9-3p表达量均高于其配对癌旁正常组织(P<0.01)。结论通过高通量测序技术发现miRNA-1228-5p、miRNA-129-5p、miRNA-760、miRNA-885-3p、miRNA-9-3p、miRNA-95-3p可能是NSCLC患者潜在的特异性miRNAs,其中miRNA-9-3p主要来源于NSCLC患者癌组织,这为后续通过外周血外泌体miRNAs测序而实现NSCLC的早期、无创诊断奠定了研究基础。

Background There is no early and effective diagnostic indicators of non-small cell lung cancer(NSCLC)at present,exosomes and their internal substances from cancer tissues can reflect the nature of parent cells,which is expected to become biological markers for the early diagnosis of tumor.Objective To screen the differentially expressed microRNAs(miRNAs)in peripheral blood exosomes in patients with by high-throughput sequencing.Methods Peripheral blood samples of 4 patients with NSCLC were collected in Shenzhen People’s Hospital from January 2018 to January 2019,including 2 patients with lung adenocarcinoma(LUAD)and 2 patients with lung squamous cell carcinoma(LUSC);meanwhile peripheral blood samples of 4 healthy volunteers were collected as control.HiSeq 2500 high-throughput sequencer was used to complete the sequencing of miRNAs,and DESeq2 software was used to screen the differentially expressed miRNAs;miRNA-9-3 p sequencing results in the 39 pairs of LUAD tissues and matched normal paracancerous tissues,45 pairs of LUSC tissues and matched normal paracancerous tissues were compared in TCGA.Results(1)High-throughput sequencing found 776 kinds of miRNAs in peripheral blood exosomes in the 4 patients with NSCLC and 238 kinds of miRNAs in peripheral blood exosomes in the 4 healthy volunteers on average.(2)A total of 6 kinds of miRNAs were identical in the 10 miRNAs with the most significant differential expression in peripheral blood exosomes between patients with LUAD and LUSC,including miRNA-1228-5 p,miRNA-129-5 p,miRNA-760,miRNA-885-3 p,miRNA-9-3 p and miRNA-95-3 p.(3)Expression quantity of miRNA-9-3 p in the peripheral blood exosomes in LUSC-1,LUSC-2,LUAD-1 and LUAD-2 was 71.51,15.61,13.99 and 16.11 TPM,respectively,while expression quantities of miRNA-9-3 p in peripheral blood exosomes in the 4 healthy volunteers were all 0.Expression quantity of miRNA-9-3 p in LUAD tissues and LUSC tissues was statistically significantly higher than that in the matched normal tissues in TCGA,respectively(P<0.01).Conclusion miRNA-1228-5 p,miRNA-129-5 p,miRNA-760,miRNA-885-3 p,miRNA-9-3 p and miRNA-95-3 p found by high-throughput sequencing may be potential specific miRNAs for patients with NSCLC,thereinto miRNA-9-3 p mainly originated from cancer tissues of patients with NSCLC,thus it may lay the foundation of researches about early and non-invasive diagnosis of NSCLC through sequencing of miRNAs in peripheral blood exosomes.