Correlation between immune lymphoid cells and plasmacytoid DCs in colon cancer

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to use RNA-seq to compare ILC3s and pDCs gene expression in colon cancer. Results: Using an optimized data analysis workflow, More than >60 million clean reads were obtained from each sample group after elimination of low-quality reads. A total of 14,943 in ILC3s and 10,840 in pDCs DEGs were found up-regulated and 4,213 in ILC3s and 11,549 DEGs in pDC s down-regulated on comparison of ILC3s and pDCs from tumor samples and controls samples transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl-/- retina, with a fold change =1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusion: These findings highlighting the important roles of ILC3s and pDCs in the processes of tumor progression and inhibition in colon cancer promote the development of new strategies for inducing antitumor immune responses in metastatic and recurrent colon cancer. Overall design: Methods: ILC3s and pDCs mRNA profiles of tumor sample and those of control were generated by deep sequencing, using Hiseq Xten.The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks.

研究目的：下一代测序（Next-generation sequencing, NGS）彻底革新了细胞通路的系统生物学分析。本研究旨在通过RNA测序（RNA-seq）对比结肠癌组织中ILC3s与pDCs的基因表达差异。研究结果：采用优化的数据分析流程，每组样本经低质量读段过滤后，均获得超过6000万条洁净读段。通过TopHat分析流程对比肿瘤样本与对照样本来源的ILC3s和pDCs的转录本后，发现ILC3s中共有14943个差异表达基因（Differentially Expressed Genes, DEGs）上调、4213个下调，pDCs中则分别有10840个上调、11549个下调。RNA测序数据证实了25个已知持家基因的稳定表达，其中12个经实时定量聚合酶链反应（qRT-PCR）验证。RNA测序数据与qRT-PCR结果在超过四个数量级范围内呈线性相关，拟合优度（R²）为0.8798。约10%的转录本在野生型（Wild Type, WT）与Nrl基因敲除（Nrl-/-）视网膜中存在差异表达，差异倍数为1.5，p值小于0.05。25个基因的表达变化经qRT-PCR验证，证实了RNA测序方法具有极高的灵敏度。对差异表达基因进行层次聚类分析后，发现了若干尚未被注释的基因，这些基因可能与视网膜功能相关。采用Burrows–Wheeler比对器（Burrows–Wheeler Aligner, BWA）与TopHat两种分析流程进行数据挖掘，结果显示二者存在显著的结果重叠，但同时也为转录组分析提供了互补的研究视角。研究结论：本研究结果揭示了ILC3s与pDCs在结肠癌肿瘤进展与抑制过程中的重要作用，可为转移性及复发性结肠癌的抗肿瘤免疫应答诱导新策略开发提供理论支撑。整体实验设计与方法：通过HiSeq Xten平台对结肠癌肿瘤样本与对照样本中的ILC3s及pDCs的mRNA进行深度测序，获取转录组数据。对通过质量过滤的序列读段，分别采用两种分析方法在转录异构体水平进行解析：一是Burrows–Wheeler比对器（BWA）结合方差分析（Analysis of Variance, ANOVA），二是TopHat结合Cufflinks。