[Targeting hTERT gene influenced the viability on hMSCs by small hairpin RNA].
OBJECTIVE
To observe the effect of shRNA by targeting human telomerase reverse transcriptase (hTERT) mRNA in the human mesenchymal stem cells (hMSCs) lines and explore the safety of RNAi by targeting hTERT mRNA in clinical application.


METHODS
According to the strategy of RNAi, which determined the specific base sequences to design shRNA plasmid. Plasmid shRNA1 involved in fluorescein gene and homologous to hTERT were designed. control shRNA2-a random sequences heterogeneous to mankind's gene were also constructed. METAFECTENE was used as the transfection reagent. The experiment was divided into 4 groups: A (shRNA1), B (shRNA2), C (metafectene) and D (normal culture medium ). At 24h, fluorescence expression was detected by confocal microscopy after administration of plasmid. At 24h, 48h and 72h cells viability were determined using the MTT assay. At 48h, the morphological change were examined by HE stain. At 48h, hTERT mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) and the telomerase activity of hMSCs were examined by PCR ELISA.


RESULTS
Many cells gave out green fluorescent after treated by shRNA1 and shRNA2. It was observed that the viability of hMSCs hadn't change after treatment with different methods within 72h (P > 0.05). Cells, morphology and density had not change after treated by different factor. All cells didn't express hTERT mRNA and the telomere activity of hMSCs was negative.


CONCLUSIONS
The results suggest that shRNA plasmid directed against hTERT can not influence the viability on hMSCs. The treatment with RNA interference may be a safety strategy for gene therapy in vivo.
Cheng
Jie
J
Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Tao
Ze-Zhang
ZZ
Xiao
Bo-Kui
BK
Duan
Hong-Gang
HG
Chen
Shi-ming
SM
D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D019098:Telomerase / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; C509186:TERT protein, human; D019098:Telomerase