Integrating metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction. Integrating metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction

Purpose: The goal of this study is to integrate NGS-derived parotid salivary gland transcriptome profiling (RNA-seq) to metabolomics (UPLC-MS/MS) methods and identify mechanisms driving radiation-induced xerostomia, with potential clinical application to head and neck cancer patients. Methods: Parotid salvary gland mRNA profiles of 28-day-old wild-type (WT) and 5 Gy radiation treated mice were generated by deep sequencing, in triplicate, using Illumina TruSeq. The sequenced reads that passed quality filters were trimmed with Trimmomatic, mapped to mm10 genome with HISAT2, and summarized at the gene level with HTSeq-count and analyzed with DESeq2. Results: Using an optimized data analysis workflow, we mapped sequenced reads per sample to the mouse genome (build mm10) and identified 25422 genes with at least 1 read in the parotid glands of WT and radiation treated mice with HISTA2 workflow. RNA-seq data QA/QC by Principal Component Analysis showed a clear separation between conditions. 155 genes showed significant differential expression between the WT and radiation treated salivary gland, with an adjsuted p-value < 0.05. Pre ranked Gene Set Enrichment Analysis against the CPDB database led to 859 altered pathways with a p-value < 0.05. Integrative analysis pointed to 103 RNA-Seq/Metabolite joint enriched pathways with a p-value < 0.05. Overall design: Parotid salivary gland mRNA profiles of 28-day old wild type (WT) and radiation treated mice

研究目的：本研究旨在整合下一代测序（Next-Generation Sequencing, NGS）来源的腮腺转录组谱分析（RNA-seq）与代谢组学（UPLC-MS/MS）方法，阐明放射性口干症的潜在发病机制，以期为头颈部癌症患者提供潜在临床应用价值。
实验方法：采用Illumina TruSeq建库试剂盒，对28日龄野生型（WT）及5戈瑞（Gy）辐射处理小鼠的腮腺mRNA进行深度测序，设置三次生物学重复。经质量过滤的测序reads通过Trimmomatic进行序列修剪，利用HISAT2比对至mm10小鼠参考基因组，通过HTSeq-count完成基因水平计数汇总，并借助DESeq2开展差异表达分析。
实验结果：通过优化后的数据分析流程，我们将每个样本的测序reads比对至构建版本为mm10的小鼠基因组，在野生型及辐射处理小鼠的腮腺组织中，共鉴定出25422个至少包含1条测序读段的基因。基于主成分分析（Principal Component Analysis）的RNA-seq数据质量控制显示，两组样本呈现清晰的分离趋势。野生型与辐射处理小鼠腮腺组织间共有155个基因存在显著差异表达（校正后P值<0.05）。针对CPDB数据库开展的预排序基因集富集分析（Gene Set Enrichment Analysis）共筛选出859个P值<0.05的异常调控通路。整合分析结果显示，共有103个RNA-seq与代谢物联合富集通路的P值<0.05。
实验整体设计：28日龄野生型（WT）与辐射处理小鼠的腮腺mRNA转录组谱