Inhibition of Human Telomerase Reverse Transcriptase in Hep-2 Cells Using Short Hairpin RNA Expression Vectors

Abstract

Telomerase activity is mainly regulated by the human telomerase reverse transcriptase (hTERT) gene. Our objective was to investigate the effect of short hairpin RNA (shRNA) on hTERT expression and telomerase activity in laryngeal cancer cells. Design: Short hairpin RNA expression vectors targeting the messenger RNA of hTERT were constructed. Cells were treated with shRNA expression vectors directed against 2 different hTERT sites, control vectors that included mismatched shRNA and those without shRNA. The expression of hTERT was determined by reversetranscriptase polymerase chain reaction and Western blotting. The activity of telomerase was measured by telomeric repeated amplification enzyme-linked immunosorbent assay. The cell viability was examined using the 3-(4,5-dimethyl thizol-2-yl) 2,5-diphenyl tetrazolium bromide assay. Results: We found that treatment of shRNA expression vectors induced a significant decrease in hTERT messenger RNA expression, the level of hTERT protein, telomerase activity, and cell viability. All of these effects were seen regardless of the target site, and the shRNA control showed none of these effects. Conclusion: Our results suggest that shRNA directed against hTERT inhibits telomerase activity through suppression of the hTERT expression in laryngeal cancer cells and that RNA interfering technology may be a promising strategy for the treatment of laryngeal cancers.

Effect of blocking VEGF, hTERT and Bcl-xl by multiple shRNA expression vectors on the human laryngeal squamous carcinoma xenograft in nude mice

“…The structure of multiple shRNAs and their vectors was as described previously. 6 Cell culture. The human laryngeal squamous carcinoma cell line (Hep-2) was purchased from the China Center for Type Culture Collection (Wuhan, China).…”

“…6,16 Apoptosis plays an essential role as a protective mechanism against carcinogenesis by eliminating genetically damaged cells, initiated cells as well as those cells that have progressed to malignancy. There are some anti-apoptotic genes in cancer cells that contribute to chemotherapy or radiotherapy resistance and can serve as RNAi targets.…”

“…Apoptosis in xenograft tumors were determined using a TUNEL kit (in situ cell apoptosis detection kit, Boehringer Mannheim, Mannheim, Germany) as described by Zhang 8 and our previous work. 6 All procedures were performed following the instructions provided by the manufacturer. Two controls per assay were performed: incubating sections with DNase I served as a positive control and omission of the terminal transferase from the reaction mixture served as a negative control.…”

“…6,7 In order to maximize the efficiency and versatility of the vector-based siRNA approach, here we constructed multiple shRNA expression vectors, pEGFP-shVEGF-shTERT-shBcl-xl, which targeted 3 different genes in cancer cells. We further characterized the effects of these vectors on human laryngeal squamous carcinoma in vivo.…”

Ingeniería genética vegetal para resistencia viral y vectores virales en genética reversa

“…En los últimos años se está aplicando la tecnología del silenciamiento de ARN mediada por hpARN para obtener plantas resistentes a virus (Di Nicola-Negri et al, 2005;Fuentes et al, 2006;Yan et al, 2007), reportándose en todos los casos que estas construcciones pueden inducir silenciamiento génico postranscripcional con casi un 100 por ciento de eficiencia cuando están dirigidas contra virus o genes endógenos (Smith et al, 2000). La transformación de plantas con construcciones ARN hairpin además ha sido usada para descubrir o validar la función de los genes , pero también son valiosas en genética reversa, genómica e ingeniería de rutas metabólicas (Smith et al, 2000;Chen et al, 2006;Sliva and Schnierle, 2010). Estas construcciones génicas que contienen casetes de expresión de repetidos invertidos son mucho mas eficientes que el silenciamiento mediado por construcciones sentido o antisentido (Hu et al, 2011).…”