KEGG enrichment analysis of differentially expressed genes in aged human lymphocytes induced by Alu antisense RNA

The lymphocytes were adjusted to 4 x106/ml using RPMI1640 (Eurobio, France) supplemented with 10 % FCS-1000 U/ml rIL-2 (Cali-Bio,USA), and treated with 10 mg/ml phytohemagglutinin (PHA, Zhengzhou Baiji Biotechnology, Co., Ltd) (PHA treatment). The aged lymphocytes were first treated with PHA for 12 d (referred to as 0 d group), and then lymphocytes were divided into 2 groups, one group was still treated with PHA activation for 6 d (PHA group), and the aother group was simultaneously transfected with Alu asRNA on the basis of PHA activation for 6 days (Alu asRNA+PHA group).

RNA-seq analysis was performed by Annuo Yoda Gene Technology (Beijing) Co., Ltd., according to a previously reported protocol. Compared with 0-day group lymphocytes, there were 356 differentially expressed genes (DEGs) in PHA group, of which 141 genes were up-regulated and 215 genes were down-regulated (Table 1). Compared with 0-day group lymphocytes, the Alu asRNA+PHA lymphocytes had 375 DEGs, of which 112 genes were up-regulated and 263 genes were down-regulated (Table 2).

In Table 1 and Table 2, lane 1 represents the Gene id of DEGs; lane 2 represents Foldchange of DEGs; lane 3 represents the p-value; lane 4 represents the down-regulated or up-regulated; lane 5 represents the descrption of the DEGs.   The gene ontology (GO) enrichment of the Top 30 upregulated DEGs in the Alu asRNA+PHA group compared with 0 d group was demonstrated using the GO Chord. As shown in Figure 1, compared with the 0 d group, among the up-regulated Top genes, there are 8 genes related to immune response, which are IFI44L, PTGDR2, TNFSF4, IFI44, TRIM22, CXCR6, CCR5 and TNFSF8.

There are four genes related to the activity of C-C chemokine receptors: CCR2, CXCR2, CCR5, and CXCR6. In Figure 1, on the left are genes, on the right are GO terms, and the lines in the middle indicate affiliations.When we performed a Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analysis. We found that compared with the 0 d group, the down-regulated Top DEGs in the Alu asRNA+PHA group mainly focused on TNF signaling pathway, MAPK signaling pathway, lipid and atherosclerosis and IL-17 signaling pathway, and also included osteoclast differentiation (figure 2A).

Upregulated Top DEGs are mainly concentrated in cytokine-cytokine receptor interactions, microRNA, NOD-like receptor signaling pathways, and chemokine signaling pathways in cancer (Figure 2B).