[Inhibitory effect of hTERT dsRNA on telomerase activity in lung carcinoma cell line A549].
OBJECTIVE
RNA interference (RNAi) is a new technology in gene study. The mechanism of RNAi is that double-stranded RNA (dsRNA) can band target mRNA and decompose it. This study was to assess possibility and specificity of dsRNA on suppressing human telomerase reverse transcriptase (hTERT) in lung carcinoma cells, investigate its effect on cell proliferation to confirm whether it has unspecific killing activity on mammalian cells, and explore its application in lung cancer research and treatment.


METHODS
Sequences of 2 exons and 1 intron of hTERT gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR) or PCR. The sense and antisense cDNA sequences were connected in a tandem manner, and the whole fragment was inserted into pCI-neo mammalian expression vector to construct the dsRNA expression vector, and then transfected into lung carcinoma cell line A549. The expression of hTERT was detected by RT-PCR and Western blot. Telomerase activity was measured by telomerase repeat amplification protocol (TRAP). Cell morphology was observed, and cell proliferation was assessed under invert microscope.


RESULTS
After transfection of 2 exon fragments of hTERT dsRNA, mRNA and protein expression of hTERT and telomerase activity in A549 cells were suppressed, cell proliferation was markedly inhibited. Meanwhile, dsRNA didn't show unspecific toxic activity on A549 cells.


CONCLUSIONS
hTERT dsRNA can specifically silent hTERT gene, inhibit telomerase activity and proliferation of A549 cells. hTERT dsRNA might be a potential method of gene therapy for lung cancer.
Tian
Feng-Jun
FJ
Department of Respiratory Medicine, The Third Hospital, Hebei Medical University, Shijiazhuang, Hebei, 050051, P.R.China.
Wang
Zhi-Yong
ZY
Ma
Jun-Yi
JY
Zhao
Yun-Xia
YX
Lu
Wei
W
D000230:Adenocarcinoma / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004268:DNA-Binding Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D019098:Telomerase