Novel miRNA Expression Cassette Downregulates Telomerase Reverse Transcriptase and Inhibits Cell Growth in Retinoblastoma Cells br

Objective The present study attempted to design novel miRNA expression cassettes (MEC) targeting specific consensus sequences of hTERT and hTR, which remedied the degradability and cytotoxicity and provided a convenient method for miRNA synthesis. Methods The MECs specific to hTERT and hTR were constructed by overlap polymerase chain reaction (PCR). Telomeric repeat amplification protocol (TRAP) -silver staining and TRAP real-time PCR analysis were used to determine the telomerase activity. The telomere length was determined by real-time PCR, whereas cell viability was determined using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell apoptosis rate and cell cycle were assessed using the annexin V/propidium iodide (PI) double staining and PI single staining assays, respectively, coupled with flow cytometry. Results The telomerase-specific MECs were successfully constructed. Each MEC inhibited the telomerase activity differently. Telomerase silencing could induce immediate growth arrest in the G0/G1 phase and led to retinoblastoma (RB) cell apoptosis. Conclusion miRNA-mediated telomerase silencing is an efficient strategy to impair RB cell growth. A robust system must be developed to fully explore the efficacy of miRNAs. The constructed MECs exhibited a strong RNAi effect and thus may be utilized to effectively screen RNAi-targeted sequences for RB gene therapy.