Differentially expressed genes in senescent human lymphocytes induced by Alu antisense RNA

Short interspersed nuclear elements (SINEs), comprising 10% of the mammalian genome, participate in aging regulation through intricate molecular networks. Canonical representatives such as human Alu and murine B1 elements, although predominantly located in intronic regions, demonstrate bidirectional regulatory effects. Crucially, Alu elements exhibit strong associations with age-related alternative splicing dysregulation, inducing pathogenic outcomes through aberrant splice site creation or premature translational termination. Our previous studies demonstrated that murine SINE B1 antisense RNA delays cellular senescence by suppressing reactive oxygen species (ROS) accumulation and modulating the expression of senescence-associated genes, establishing SINE elements as a potential therapeutic target for anti-aging interventions. Here, we investigated the changes in the gene expression profile of lymphocytes separated from senescent individuals (more than 60 year old) treated with Alu antisense RNA (Alu asRNA). The lymphocytes were resuspended at 1´106 cells/mL in complete RPMI 1640 medium and seeded into 96-well plates (125 μL/well). The lymphocytes were activated with phytohemagglutinin (PHA, 10 μg/mL) (Ningbo Tianan Biologic Material Co., Ltd.) and recombinant interleukin-2 (rIL-2, 1000 U/mL) (Beijing SL Pharmaceutical Co., Ltd.). For calcium phosphate transfection (CPT), a transfection complex was prepared by mixing 1 mg/mL Alu asRNA with 0.1 M CaCl2, followed by dropwise addition to 2 ´HBS (HEPES-buffered saline) to form Alu asRNA-calcium phosphate precipitates. The mixture was then added to cultured lymphocytes. The sequence of the Alu asRNA was: TGAGACGGAGTCTCGCTGTGTCGCCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACTGCAAGCTCCACCTCCCAGGTTCACGCCATTCTCCTGCCTCAGCCTCTTGAGTAGCTGGGACTACAGGCACCCGCCACCACACCCGGCTAATTTTTTTGCATTTTTAGTAGAGACGGGGTTTCACCGTATTAGCCAGGATGGTCTTGATCTCCTGACCTTGTGATCCGCCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCC. The transfected lymphocytes were cultured at 37℃ with 5% CO2 for 6 d. Lymphocytes treated only with PHA and rIL2 were taken as the control group; Lymphocytes transfected with Alu asRNA on the basis of PHA and rIL2 treatment were the experimental group (Alu asRNA group). RNA-seq analysis was performed by Shanghai OE Biotech. Co., Ltd., according to a previously reported protocol. The results showed that 136 differentially expressed genes (DEGs) (Fold change ≥1.5, P<0.05), including 89 up-regulated genes and 47 down-regulated genes in Alu asRNA group compared with control group. DEGs were shown in Table 1. Lane 1 represents the Gene id of DEGs; lane 2 represents Foldchange of DEGs; lane 3 represents the p-value; lane 4 represents the doen-regulated or up-regulated; lane 5 represents the descrption of the DEGs.