[Effects of hTERT RNAi on apoptosis of hepatocellular carcinoma cells induced by TRAIL].
OBJECTIVE
To study the effects of human telomerase reverse transcriptase (hTERT) RNA interference (RNAi) on biological characteristics of hepatocellular carcinoma cell lines HepG2 and SMMC-7721 and on apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).


METHODS
Small hairpin hTERT (shTERT) sequence was identified by PCR method; hTERT expressions, morphological features, cell proliferation and replicative senescence were respectively determined using RT-PCR, hematoxylin-eosin (HE) staining, growth curve and beta-galactosidase (b-Gal) staining; cell cycle and apoptosis were identified using flow cytometry after propidium iodide (PI) staining and annexin V/PI double staining.


RESULTS
shRNA were found in 6/8 HepG2 and 6/6 SMMC-7721 cell clones transformed by the recombined plasmid pSilencer 3.1-H1 neo-shTERT. The interference rates of hTERT on HepG2 and SMMC-7721 were 100% and 43.3% respectively. Cells in G2-M phases increased from 7.1% to 10.6% and from 6.9% to 7.9% respectively; and the percentage of replicative senenscence cells increased from 0 to 20.4% and from 3.6% to 10.0% respectively. The nucleus/cytoplasm ratios of the cells were obviously decreased after hTERT RNAi treatment. Moreover, apoptosis of hepatocellular carcinoma cells and apoptosis induced by TRAIL were strikingly increased by hTERT RNAi (P < 0.05). For example, apoptosis rates were increased from 3.5% to 5.2% in HepG2 cells and from 4.8% to 7.9% in SMMC-7721 cells after hTERT RNAi treatment. Apoptosis rates were increased from 5.3% to 10.4% in HepG 2 cells and from 13.9% to 77.2% in SMMC-7721 cells after being treated by 100 ng/ml TRAIL for 24 h. However, there were no remarkable changes between control cells and untransformed cells.


CONCLUSIONS
hTERT RNAi not only has a significant effect on biological characteristics of hepatocellular carcinoma cells, but also obviously can increase cell apoptosis induced by TRAIL.
Zhang
Ru-gang
RG
Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Fang
Dian-chun
DC
Luo
Yuan-hui
YH
Guo
Li-ping
LP
D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D019098:Telomerase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; C509186:TERT protein, human; D019098:Telomerase