[Inhibition of growth and proliferation of Hep-2 cells by targeting human telomerase reverse transcriptase mRNA using RNA interference technology].
OBJECTIVE
To investigate the effect of RNA interference by targeting human telomerase reverse transcriptase (hTERT) mRNA in the larynx cancer cell line, Hep-2.


METHODS
The primary structures of hTERT cDNA were found in GenBank. Then the structure analysis were done according to RNAi strategy which determined the specific base sequences to design shRNA plasmid. Two types of plasmid, pshRNA1 and pshRNA2, involved in fluorescein gene were synthesized based on the specific base sequences. Control pshRNA3, a random sequence, and control pshRNA4, without additional specific sequence were also constructed. Cells were treated daily with pshRNA1-4 or normal culture medium respectively. The pshRNA1-3 was identified by electrophoresis. After administration of pshRNA1-4, fluorescence expression was detected by confocal microscopy, the expression of hTERT of the transfected cells was determined by Western blotting, telomerase activity was measured by TRAP-PCR ELISA, cell viability was determined by MTT assay, morphological changes and apoptosis were examined by inverted microscope and TUNEL respectively.


RESULTS
There was a 400 bp balteum in pshRNA1-3 after cut by SalI, which was identical with the size of the objective gene. Many cells presented green fluorescence after being treated by pshRNA1-4, but there are much more dead green fluorescent cells in the pshRNA1 and pshRNA2 group. hTERT protein and telomerase activity was significantly decreased after treated by pshRNA1 or pshRNA2. It was observed that treatment with pshRNA1 or pshRNA2 in the presence of a valid transfection reagent could reduce cell viability of Hep-2 cells within 96 h (P < 0.01). Under the same culture conditions, cells grew more sparsely and the number of apoptotic cell increased significantly.


CONCLUSIONS
shRNA plasmid directed against human telomerase reverse transcriptase can effectively transfect Hep-2 cells. shRNA targeted hTERT gene can significantly inhibit the growth and proliferation of Hep-2 cells, which results in apoptotic cell death. RNA interference may be a promising strategy for the treatment of laryngeal cancer.
Chen
Shi-ming
SM
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Tao
Ze-zhang
ZZ
Xiao
Bo-kui
BK
Pan
Song
S
Liu
Dan
D
Chi
Hua-ming
HM
D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D007822:Laryngeal Neoplasms / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019098:Telomerase