[Inhibitory effect of silencing hTERT gene by short hairpin RNA on growth of human laryngeal squamous cell carcinoma xenograft in nude mice].
OBJECTIVE
RNA interference (RNAi) is triggered by the presence of double-stranded RNA (dsRNA) in the cell and results in rapid destruction and post-transcriptional gene silencing of target mRNA. The roles of RNAi in gene function and antiviral therapy have been reported, but its effect on human telomerase reverse transcriptase (hTERT) gene, which is highly expressed in laryngeal squamous cell carcinoma and other head and neck neoplasms, has seldom been reported. This study was to explore the inhibitory effect of silencing hTERT gene by short hairpin RNA (shRNA) on growth of laryngeal squamous cell carcinoma xenograft in nude mice with RNAi technique.


METHODS
Plasmid pshRNA containing fluorescein gene and hTERT cDNA sequences was synthesized. Human laryngeal squamous cell carcinoma Hep-2 cells were transplanted into nude mice to establish xenograft tumors. pshRNA was transfected into the tumors. The tumor volume was observed. Fluorescence expression in the tumors was observed by laser confocal microscopy. Tumor morphology was observed with HE staining. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The expression of hTERT protein in the tumors was detected by SP immunohistochemistry. The structural change of heart, liver, kidney and spleen was observed, and peripheral blood hematological and biochemical parameters were determined.


RESULTS
The tumor volume was significantly smaller in pshRNA group than in normal saline (NS) group and blank plasmid group (P<0.01). The inhibition rate was 76.50% in pshRNA group. After transfection of pshRNA or blank plasmid, many green fluorescent cells were observed under confocal microscope. The necrosis and apoptosis of tumor cells were found under light microscope in shRNA group. The apoptotic index of tumor cells was significantly higher in pshRNA group than in NS group and blank plasmid group [(26.47+/-4.25)% vs. (2.73+/-1.35)% and (3.40+/-1.41)%, P<0.05]. pshRNA directly down-regulated hTERT protein expression in the tumors, but did no damage to the heart, liver, kidney, spleen, and blood system of nude mice.


CONCLUSIONS
shRNA plasmid containing specific sequences of hTERT gene could significantly inhibit the growth of laryngeal carcinoma in nude mice, with no side effect on the heart, liver, kidney, spleen, and blood system.
Liu
Dan
D
Department of Otolaryngology-Head and Neck Surgery, People's Hospital, Wuhan University, Wuhan, Hubei 430060, P. R. China.
Tao
Ze-Zhang
ZZ
Xiao
Bo-Kui
BK
Chen
Shi-Ming
SM
Chi
Hua-Ming
HM
D000818:Animals; D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010957:Plasmids; D034741:RNA, Small Interfering; D011897:Random Allocation; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D019098:Telomerase