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Abstract
Background:
We have determined the effects of human telomerase RNA inhibiton using siRNA in tumor cells and human embryonic and mesenchymal stem cells.
Methods:
We selected the sequences against the predicted loop; these sequneces were comprised of nucleotides from 76 to 94 residues and from 143 to 163 residues as the target sequences, and we cloned these sequences into pU6sh75 and pU6sh143 cells. Three different kinds of cell lines were used: HeLa, SNUhES3, and human mesenchymal stem cells. The degree of inhibition of telomerase activity was assessed by TRAP assay and RT-PCR.
Results:
The telomerase activity of the HeLa and SNUhES3 cells were 135.3±14.5 and 109.0±18.2; these cells showed higher activity than human mesenchymal stem cells and Wi38 cells (46.3±5.0 and 26.0±12.0), which were control cells. When each of the types of cells was treated with siRNA-hTR, the transfection efficiency of pU6sh75 for the HeLa, SNUhES3, and human mesenchymal stem cells was 91.0±8.4%, 83.3±16.0% and 81.9±12.3%, respectively. In the case of pU6sh143, its transfection efficiency was similar to pU6sh75; the HeLa, SNUhES3 and human mesenchymal stem cells tranfection efficiency was 90.1±9.0%, 79.9±18.2% and 79.4±15.1%, respectively. After two days of transfection, the level of telomerase activity in the pU6sh75 transfected cells decreased to 64.3±10.1% and 56.0±11.0% in the HeLa and SNUhES3 cells, respectively. When the cells were transfected with pU6sh143, the telomerase activity also decreased in the HeLa and SNUhES3 cells (71.3±9.1% and 61.6±8.3%, respectively). However, the difference of telomerase activity was not significant in the human mesenchymal stem cells: 43.0±7.2% with pU6sh75 and 46.0±9.0% with pU6sh143.
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Fig. 1
Sequences and predicted secondary structure of hairpin RNAs derived from the U6 promoter-driven expression constructs. Mutant versions of each siRNA with 3 points mutation at the sequences of the target were also shown.
Fig. 2
Telomerase activity in experimental cell lines. The telomerase activity was assayed by a telomere repeat amplification protocol (TRAP) assay using 2,000 cells. The telomerase activity of the U293 cell line was considered as 100, with the telomerase activity of each cell line normalized to this. The means from at least three independent experiments are shown.
Fig. 3
Inhibition of Telomerase activity by siRNA. The effects of siRNAs are expressed as the percentage of the telomerase activity compared with mutant versions (hTR75 m, hTR143m). The means from at least three independent experiments are shown.
Fig. 4
Inhibition of mRNA expression of hTR. In the siRNA-hTR143 transfected ES, mRNA expression of hTR was decreased compared to control ES and siRNA-hTR143m transfected ES. M, marker for molecular weight; lane 1 and 4, control ES; lane 2 and 5, siRNA hTR143m transfected ES; lane 3 and 6, siRNA hTR143 transfected ES.
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