Single-cell RNA sequencing for the identification of early-stage lung cancer biomarkers from circulating blood

To develop biomarkers for diagnosis of early-stage lung adenocarcinoma (LUAD) patients, we set up single-cell RNA sequencing (scRNA-seq) pipeline using Fluidigm C1 systems. Through the scRNA-seq pipeline, we clustered 1,441 single cells from four non-small cell lung cancer (NSCLC) cell lines (A549, H460, H1299 and Calu3) into four groups based on differential gene expression and detected over 2,400 differentially expressed genes (DEGs) from a comparison of four clusters following dimension reduction. We validated fold change values of 120 DEGs selected from three cluster comparisons (top 20 up- and top 20 down-regulated genes per cluster comparison) using qRT-PCR approach. We performed further quantitative validation of chemokine genes (CXCL1 and CXCL2 with corresponding receptor, CXCR2) at mRNA level in tumour and tumour-adjacent normal lung tissues of LUAD patients (16 female and 19 male). Next, we profiled microRNAs epigenetically regulating expression of those chemokine genes in the tumour and normal tissues as well as blood (plasma) samples from corresponding LUAD patients. Finally, we identified relatively high copy number of miR-532-5p regulating those chemokine genes in patient plasma samples. We loaded 400 single cells per NSCLC cell lines (A549, H460, H1299 and Calu3) to chambers of high-throughput (HT) scRNA-seq integrated fluidics circuits (IFCs). We generated four hundreds of 3'-end enriched scRNA-seq libraries per NSCLC cell line using in Fluidigm C1 systems (1,600 libraries in total). Those libraries were sequenced using illumina Nextseq 500 systems.

为开发早期肺腺癌（lung adenocarcinoma, LUAD）患者的诊断生物标志物，本研究搭建了基于Fluidigm C1系统的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）流程。通过该scRNA-seq流程，我们对源自4株非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞系（A549、H460、H1299及Calu3）的1441个单细胞进行聚类，基于差异基因表达模式将其划分为4个细胞群；经降维分析后，对比4个细胞群的转录组特征，共检测到2400余个差异表达基因（differentially expressed genes, DEGs）。我们选取3组细胞群对比中筛选出的120个DEGs（每组细胞群对比选取排名前20的上调基因与前20的下调基因），采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）对其表达倍数变化值进行验证。我们进一步在16例女性、19例男性LUAD患者的肿瘤组织与肿瘤旁正常肺组织中，对趋化因子基因CXCL1、CXCL2及其受体CXCR2的信使RNA（messenger RNA, mRNA）水平开展定量验证。随后，我们在上述LUAD患者的肿瘤组织、正常肺组织及血液（血浆）样本中，分析了表观遗传调控上述趋化因子基因表达的微小RNA（microRNAs, miRNAs）的表达谱。最终，我们在患者血浆样本中鉴定出可调控上述趋化因子基因的高拷贝数miR-532-5p。此外，我们向高通量（high-throughput, HT）单细胞RNA测序集成流体回路（integrated fluidics circuits, IFCs）的反应腔室中，为每株NSCLC细胞系接种400个单细胞；基于Fluidigm C1系统，我们为每株NSCLC细胞系构建了400个3'端富集型单细胞RNA测序文库，总计1600个文库，并采用Illumina Nextseq 500测序平台对这些文库进行测序。