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

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.