The splicing factor hnRNP M is a critical regulator for innate immune gene expression in macrophages

Purpose: The goals of this study are to compare hnRNP M shRNA knockdown macrophages to scramble shRNA control macrophages in uninfected cells and Salmonella Typhimurium-infecetd cells to unbiasly look at gene expression changes that are regulated by the splicing factor, hnRNP M during resting state and during an innate immune response. Methods: Macrophage mRNA profiles of uninfected and Salmonella Typhimurium-infected hnRNP M knockdown cells lines and SCR shRNA control RAW264.7 macrophages were generated by sequencing, in triplicate, using Illumina 1.9 system. The sequence reads that passed quality filters were analyzed at the gene expression level with CLC Genomics Workbench 8 with Transcriptomeics Analysis followed by statistical analysi with Empiciral Analysis of DGE and (EDGE test) and Baggerly's test. qRT–PCR validation was performed using SYBR Green assays. Results: Using CLC Genomics Workbench 8 transcriptomics workflow, we mapped about 30 million sequence reads per sample to the mouse genome (GRCm38). Approximately 140 transcripts showed differential expression between the scramble control and hnRNP M knockdown macrophages in uninfected cells, with a fold change =1.5 and p value <0.05. Additionally, ~150 transcripts showed differential expression between the scramble control and hnRNP M knockdown macrophages in Salmonella-Typhimurium cells, with a fold change =1.5 and p value <0.05. Altered expression of genes was confirmed with qRT–PCR, demonstrating the effectiveness of the RNA-seq method. Conclusions: Our study demonstrates hnRNP M-dependent differential gene expression in the context of the innate immune response. Overall design: Transcriptome profiles of uninfected and Salmonella Typhimurium-infected hnRNP M shRNA knockdown and scramble control shRNA knockdown RAW 264.7 macrophages were generated by sequencing, in triplicate, using Illumina 1.9.

研究目的：本研究旨在对比未感染细胞及鼠伤寒沙门氏菌（Salmonella Typhimurium）感染细胞中，异质核糖核蛋白M（heterogeneous nuclear ribonucleoprotein M，hnRNP M）短发夹RNA（short hairpin RNA，shRNA）敲低巨噬细胞与阴性对照scrambled shRNA巨噬细胞的基因表达谱，以无偏倚地探究剪接因子hnRNP M在静息状态与先天免疫应答过程中调控的基因表达变化。
实验方法：本研究采用Illumina 1.9测序平台，对3次生物学重复的未感染及鼠伤寒沙门氏菌感染的hnRNP M敲低RAW264.7巨噬细胞系与阴性对照（SCR shRNA）RAW264.7巨噬细胞的mRNA表达谱进行测序。通过质量过滤的测序读段首先借助CLC Genomics Workbench 8开展转录组学分析，随后采用差异基因表达（Differential Gene Expression，DGE）经验分析、EDGE检验与Baggerly检验进行统计学分析。采用SYBR Green荧光染料法完成qRT-PCR验证实验。
实验结果：借助CLC Genomics Workbench 8的转录组分析流程，本研究将每个样本约3000万条测序读段比对至小鼠基因组（GRCm38）。在未感染巨噬细胞中，阴性对照与hnRNP M敲低组间共存在约140个差异表达转录本，倍数变化≥1.5且P值<0.05；在鼠伤寒沙门氏菌感染的巨噬细胞中，两组间共存在约150个差异表达转录本，同样满足倍数变化≥1.5且P值<0.05。qRT-PCR验证结果证实了基因表达差异，证明了RNA-seq测序方法的有效性。
研究结论：本研究证实，在先天免疫应答过程中存在依赖于hnRNP M的差异基因表达调控。
整体实验设计：本研究采用Illumina 1.9测序平台，对3次生物学重复的未感染及鼠伤寒沙门氏菌感染的hnRNP M shRNA敲低与阴性对照shRNA敲低RAW264.7巨噬细胞的转录组表达谱进行测序。