[Inhibitory effect of RNAi targeting human telomerase reverse transcriptase against human hepatocellular carcinoma cells].
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 hepatocellular carcinoma cells.


METHODS
The sequence of the siRNA targeting hTERT, U6 promoter and EGFP gene were amplified by PCR and inserted into the mammalian retroviral expression vector pLXSN to construct the recombinant retroviral vector pLXSN-EGFP-U6-siTERT. The vector was then used to infect human hepatocellular carcinoma 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 MTT assay.


RESULTS
Sequence analysis and restriction enzyme digestion showed confirmed successful construction of the recombinant expression vector pLXSN-EGFP-U6-siTERT. The telomerase activity of the infected HepG2 cells was reduced by 23.84%, 58.03% and 85.01% at 24, 48 and 72 h after the infection, respectively (P<0.05). The cell apoptosis rate of the infected cells was 29.05% at 24 h after the infection. The cell proliferation was markedly inhibited by the infection with the vector in comparison to that of the control group.


CONCLUSIONS
hTERT siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells.
Li
Hua
H
Liver Transplantation Center, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. lihua100@yeah.net
Wang
Xin-lu
XL
Yang
Yang
Y
Zhang
Jian
J
Wang
Gen-shu
GS
Jiang
Nan
N
Chen
Gui-hua
GH