SiRNA Targeting HTERT Gene Effectively Inhibits The Growth Of Larygeal Squamous Cell Carcinoma Cell Lines (Hep-2)

Aim:To investigate the effect of small interfering RNA (siRNA) targeting hTERT genein Hep-2 cells.Methods:The length of hTERT mRNA was fund from the Genebank. Beginning with theAUG start codon, with recording the AA and downstream 19 nucleotides and comparethe sequence to the appropriate genome database to eliminate any sequences withsignificant homology to other genes. In this study, we selected three target sequencesfor testing and controlled the GC content between 30%-65%, because earlyexperiments suggest that target sequences with a low GC content may be moresusceptible to siRNA-induced degradation. According to the target sequences wedesigned their sense and antisense oligonucleotide templates. In vitro cultured Hep-2cells were transfected with lipofectamine 2000, twenty-four hours after transfectionwe collected and observed the cells under the Fluorescence microscope to determinewhether siRNA was successfully transfected into the cells. Forty-eight hours latter, theinhibitory effect was detected by reverse transcriptase (RT-PCR), MTT andmorphology assay.Results:(1) siRNA are transfected into Hep-2 cells successfully;(2) The result of RT-PCR showed hTERT at mRNA level, the exogenous hTERTis knocked down 70% by siRNA.(3) The morphology and the MTT result show the hTERT SiRNA to be ableeffectively to suppress the Hep-2 cell multiplication, the suppression rate achieves65%.Conclusion:(1) We adopt bioinformatics technique to design effective target siRNA;(2) Effective hTERT siRNA can specifically degrade hTERT protein expressionby degraded hTERT mRNA level; (3) Effective hTERT siRNA can induce apoptosis and inhibitcell-proliferation. Subsequently it may inhibit tumor growth.