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

Alu elements are the most common short interspersed nuclear elements in the human genome, which account for approximately 10% of the human genome. Alu elements are transcribed by RNA polymerase III. Alu RNA expression increases under oxidative stress, virus infection, and during the aging process. The gene expression of lymphocytes in the aged individuals is changed and the immune functions of aged persons are impaired. Here, we investigated the effects of Alu antisense RNA (Alu asRNA) on gene expression of lymphocytes of aged individuals using RNA sequencing (RNA-seq) analysis. Peripheral blood lymphocytes were isolated from aged individuals (> 60 years old). 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).