RNA-seq analysis of HT8 cells based on PPDPF knockdown. RNA-seq analysis of HT8 cells based on PPDPF knockdown

Purpose: Analysis of the regulatory network involved in PPDPF in colon cancer cells. Methods: mRNA profiles of HT8-cas1 and HT8 group were generated by deep sequencing, using Illumina Novaseq platform. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate .Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed. Clusterprofiler software was used to perform GO function enrichment analysis and KEGG pathway enrichment analysis of the differential gene sets. Results: We identified 3233 differential genes, of which 1877 were up-regulated and 1356 were down-regulated. In GO enrichment analysis,the biological process is mainly in the cell ions homeostasis and the molecular function is mainly in viral life cycle,response to virus,neutrophil activation, and the transmembrane signaling receptor activity. KEGG analysis shows that differential genes are enriched in Epstein-Barr virus infection ,Glutathione metabolism and Proteasome pathways. Conclusion: Based on RNA-seq analysis, the regulatory network involved in PPDPF in colon cancer . Overall design: mRNA profiles of HT8-cas1 and HT8 group

研究目的：分析结肠癌细胞中PPDPF参与的调控网络。
研究方法：采用Illumina Novaseq测序平台进行深度测序，获取HT8-cas1组与HT8组的mRNA表达谱。使用Hisat2 v2.0.5构建参考基因组索引，并通过该软件将双端clean reads比对至参考基因组；使用featureCounts v1.5.0-p3统计比对至每个基因的reads数目，并基于基因长度与比对reads数计算各基因的FPKM值。利用DESeq2 R包（版本1.16.1）对两组样本（每组设置2个生物学重复）开展差异表达分析，所得P值通过Benjamini与Hochberg方法校正以控制错误发现率，将校正后P值<0.05的基因定义为差异表达基因。使用Clusterprofiler软件对差异基因集进行GO功能富集分析与KEGG通路富集分析。
研究结果：本研究共鉴定出3233个差异表达基因，其中1877个上调、1356个下调。GO功能富集分析结果显示，差异基因在生物学过程中主要富集于细胞离子稳态，在分子功能中主要富集于病毒生命周期、病毒应答、中性粒细胞活化及跨膜信号受体活性。KEGG通路富集分析结果表明，差异基因富集于EB病毒感染、谷胱甘肽代谢与蛋白酶体通路。
研究结论：本研究基于RNA-seq测序分析，明确了结肠癌细胞中PPDPF所参与的调控网络。
实验设计：HT8-cas1组与HT8组的mRNA表达谱