To investigate the regulation of exogenous human telomerase reverse transcriptase (hTERT) expression by the exogenous interleukin-6 (IL-6) promoter and its effect on cell senescence.

The IL-6 promoter was fused with enhanced green fluorescent protein (EGFP) (GV3-IL-EGFP) or hTERT-EGFP (GV3-IL-hT-EGFP) to construct lentiviral vectors. The corresponding virus particles were collected and transfected into the fifth generation human foreskin fibroblasts-1 (HFF-1). The replicative cell senescence model was constructed by natural passage cells, and HFF-1 transfected with recombinant virus of GV3-IL-EGFP was used as the control group and HFF-1 transfected with recombinant virus of GV3-IL-hT-EGFP was used as the gene therapy group. The expression level of hTERT protein in cells of the two groups was detected when the cells were approaching complete senescence (19th and 25th generations), and the curve of cell population doubling level (PDL) was drawn. The expression levels of radiation sensitive protein 51 (RAD51) and gamma-H2A histone family member X (γ-H2AX) proteins were detected when the growth rates of cells in the two groups were different (13th, 14th and 15th generations). The expression levels of IL-6 and IL-1α proteins were detected at the 15th, 17th and 19th generations. At the same time, the protein expression levels of RAD51 and γ-H2AX were compared between the two groups after 100 nM doxorubicin treatment for 48 h in the 13th, 14th and 15th generations.

The expression level of hTERT protein at the 19th generation in the control group and the 19th and 25th generations in the gene therapy group was significantly different (F = 941.781, P < 0.001). Compared with the 19th generation in the control group, the expression level of hTERT protein at the 19th and 25th generations in the gene therapy group were increased significantly (both P < 0.001). The protein expression levels of IL-6 (t = 61.570, P < 0.001; t = 36.527, P < 0.001) and IL-1α (t = 11.984, P < 0.001; t = 18.622, P < 0.001) at 17th and 19th generations in the gene therapy group were obviously down-regulated as compared with the control group. The expression levels of RAD51 and γ-H2AX proteins of the control group at the 14th generation (t = 3.101, P = 0.036; t = 10.226, P = 0.001) and at the 15th generation (t = 8.683, P = 0.001; t = 7.229, P = 0.002) were much higher than those of the gene therapy group. PDL curve showed that at the 19th generation, the cells in the control group stopped proliferating and were in a completely senescent state, while the cells in the gene therapy group still had proliferative ability (t = 6.856, P = 0.002). After doxorubicin treatment, the expression levels of RAD51 (t = 23.479, P < 0.001; t = 9.619, P = 0.001; t = 14.971, P < 0.001) and γ-H2AX (t = 4.275, P = 0.013; t = 6.787, P = 0.002; t = 9.569, P = 0.001) in the gene therapy group at the 13th, 14th and 15th generations were much lower than those in the control group.

During replicative cellular senescence and doxorubicin-induced cellular senescence, GV3-IL-hT-EGFP fibroblasts can reduce the accumulation of DNA damage and the levels of inflammatory factors by regulating the expression of hTERT after gene therapy.