Inhibition of telomerase RNA (hTR) in cervical cancer by adenovirus-delivered siRNA

Abstract

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. In this study, a 67-bp oligonucleotide encoding human telomerase RNA (hTR) was introduced into pSIREN, a shuttle vector for construction of recombinant adenovirus. Then the U6-RNA promoter and siRNA-encoding insert were cut out from the pSIREN and subcloned into pAdeno-X to construct the plasmid pAd-hTR. After the pAd-hTR was transfected into a mammalian cell line HEK-293, adenovirus carrying the hTR-targeting siRNA (Ad-hTR-siRNA) was obtained. We performed a series of experiments to demonstrate silencing of the telomerase mediated by Ad-hTR-siRNA in HeLa cells. Compared with control virus (Ad-NT-siRNA), Ad-hTR-siRNA significantly reduced both hTR mRNA level (by 70.21%) and telomerase activity (by 58.87%) in HeLa cells. Moreover, it induced apoptosis in HeLa cells. Treatment of subcutaneous tumor xenografted with Ad-hTR-siRNA could slow down tumor growth, at least partially due to the induction of apoptosis (Po0.05) in vivo. Taken together, our results demonstrated efficient and specific knockdown of telomerase in HeLa cell line by the hTR siRNA, and indicated the prospect of applying this siRNA expressing recombinant adenovirus system in cancer gene therapy.

Telomerase inhibition strategies by siRNAs against either hTR or hTERT in oral squamous cell carcinoma

“…The recombinant adenovirus Ad-hTR-si, which targets the template sequence 5 0 -TTGTCTAA CCCTAACTGAG-3 0 in hTR gene (GenBank number U86046) and the negative control recombinant adenovirus Ad-control, which included the negative control siRNA oligonucleotides, had been successfully prepared previously. 8 …”

“…The primers for the amplification of the internal control, glyceraldehyde-3-phosphate dehydrogenase, were listed in previous publication. 8 The program for PCR amplification was as follows: heat-activation of the enzyme at 95 1C for 10 s, 40 cycles of 95 1C for 5 s and 61 1C for 31 s (data collection), followed by a 10-min final extension at 72 1C. The relative amount of hTERT and hTR mRNA compared with the mRNA level of glyceraldehyde-3-phosphate dehydrogenase was calculated using the 2 ÀDDCt method.…”

“…Our previous study showed that the hTR-targeting RNAi recombinant adenovirus (Ad-hTR-si) possessed both in vitro and in vivo antitumor activity when it was used to infect Hela cells. 8 In addition, another group reported that simultaneously silencing multiple oncogenes in tumor cells could result in a stronger inhibition of tumor cell proliferation and induction of cell apoptosis. 9 Furthermore, a recent study revealed that hTR and hTERT function in an interdependent manner.…”

“…Half of the tumor tissue was separated, fixed with 4% paraformaldehyde, embedded in paraffin and sliced for the detection of tumor cell apoptosis by the terminal deoxynucleotidyl transferase-mediated-dUTP nick-endlabeling (TUNEL) assay (Roche, Indianapolis, IN). 8 In each case, five individual  400 visual field were analyzed for calculating the apoptosis index, apoptosis index ¼ (TUNEL staining positive cells/tumor cells)  100%. The protein expression of vascular endothelial growth factor (VEGF), which served as a marker of angiogenesis, was measured by immunohistochemistry using a VEGF antibody (Santa Cruz, Santa Cruz, CA).…”

Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells

“…Maximum value of absorbance for negative control should be 0.25, and the value should be higher than 1.5 for positive control. Samples were regarded as telomerase positive if the difference in absorbance (DA) was 0.2 higher than negative control [25].…”