Inhibitory effect of RNA interference targeting human telomerase reverse transcriptase against hepatoblastoma cells

Abstract

Objective To construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatoblastoma cells. Methods The sequences of the siRNA targeting hTERT, U6 promoter and enhanced green fluorescent protein (EGFP) gene were amplified by polymerase chain reaction (PCR) and inserted into the mammalian retroviral expression vector pLXSN. The PCR method was used to amplify hTERT-siRNA, EGFP, DNA fragment of U6+ 27. The retroviral expression vector pLXSN-EGFP-U6-siTERT was constructed and subjected to enzyme digestion identification. The recombinant retroviral vector pLXSN-EGFP-U6-siTERT was constructed. The vector was then used to infect human hepatoblastoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay. Results hTERT-siRNA retroviral expression vectors were successfully prepared. In the control group, the telomerase activity was 2 143.06±198.69. At 24, 48 and 72 h after colony forming units (CFU) recombinant virus infection the telomerase activity was 1 632.02±116.28, 899.38±126.11 and 321.25±25.25 respectively. As compared with before infection, the telomerase activity was respectively decreased by 23.84%, 58.03% and 85.01%. There was significant difference among different time groups (P=0.046, 0.024 and 0.008). The results of flow cytometry showed that there was significant difference in the cell apoptosis rate between the experimental group and the control group with different titer of recombinant virus infection, and there was a concentration-dependent relationship. At 24 h after 1×105 CFU recombinant virus infection, the apoptosis rate was 29.05%. MTT results showed that apoptosis rate was 29.05% at 24 h after recombinant virus infection, the tumor cell death was significant, and with the increase in virus titer and prolongation of time, cell death also gradually increased. At 24, 48 and 72 h after virus infection of 6.0×105 CFU, the results of MTT assay were 0.29±0.14, 0.20±0.13 and 0.18±0.11 respectively. At 24, 48 and 72 h after virus infection of 3.0×105 CFU, MTT results were 0.32±0.11, 0.26±0.12 and 0.25±0.10. At 24, 48 and 72 h after virus infection of 1.0×105 CFU, results of MTT assay were 0.33±0.12, 0.26±0.13 and 0.26±0.12. At 24, 48 and 72 h after virus infection of 1.0×104 CFU, the results of MTT assay were 0.43±0.14, 0.35±0.10 and 0.33±0.15. At 24, 48 and 72 h after virus infection of 1.0×103 CFU, the results of MTT assay were 0.52±0.11, 0.44±0.13 and 0.44±0.10. At 24, 48 and 72 h after virus infection of 1.0×102 CFU, the results of MTT assay were 0.65±0.13, 0.61±0.15 and 0.60±0.16. In the negative control group, the results of MTT assay were 0.69±0.11, 1.01±0.14 and 2.98±0.16 respectively at 24, 48 and 72 h. There existed the concentration-effect and time-effect relationship (P=0.037, 0.034 and 0.028). Conclusion hTERT-siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells. Key words: RNA interference; Human telomerase reverse transcriptase; Hepatoblastoma; Apoptosis