[A DNA vector-based RNAi technology to inhibit the activity of the telomerase of cell line HCCLM3].
OBJECTIVE
To develop a DNA vector-based RNA interference (RNAi) technology that inhibits the activity of the telomerase of the hepatocellular carcinoma cell line HCCLM3 in order to suppress the proliferation of the cells.


METHODS
Hepatocellular carcinoma cells of the line HCCLM3 were cultured. mRNA interfering double-stranded DNA vector PSG-AS targeting the mRNA of human telomerase reverse transcriptase (hTERT) and the control vector PSG-CTR were constructed respectively, and then were transfected into the HCCLM3 cells. The expression of hTERT of the transfected cells was determined by Western blotting and the activity of telomerase was determined by telomeric repeat amplification-ELISA (TRAP-ELISA). Flow cytometry was used to detect the apoptosis of transfected cells and MTS method was used to measure the growth curve of the cells so as to observe the effect of the PSG-AS on the proliferation of HCCLM3 cell.


RESULTS
TRAP-ELISA showed that the inhibition rate of PSG-AS on the telomerase activity was 76%. The apoptotic rate of the PSG-AS group was significantly higher than that of the PSG-CTR group (t = 11.48, P < 0.001). Western blotting showed a remarkable inhibition of hTERT protein in the PSG-AS group.


CONCLUSIONS
Capable of suppressing the hTERT expression and the activity of telomerase, RNA interfering technology can be applied to treatment of tumors.
Lu
Xiao-dong
XD
Institute of Life Science, Jiangsu University, Zhenjiang 212013, China.
Qin
Wen-xin
WX
Pan
Dong-ning
DN
Li
Jin-jun
JJ
Wan
Da-fang
DF
Wen
Chuan-jun
CJ
Li
Chao-jun
CJ
Gu
Jian-ren
JR
Yang
Sheng-li
SL
D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D002455:Cell Division; D045744:Cell Line, Tumor; D004260:DNA Repair; D004268:DNA-Binding Proteins; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D019098:Telomerase