title: Targeting Human Telomerase Reverse Transcriptase by a Simple siRNA Expression Cassette in HepG2 Cells.
abstract: BACKGROUND
  Human telomerase reverse transcriptase (hTERT) has become an ideal target for development of anticancer therapy. Small interfering RNAs (siRNAs) are very powerful reagents for gene silencing and show promise for cancer gene therapy. However, only a small number of siRNAs have been demonstrated to be effective. For gene therapy targeting hTERT, it is essential to develop a robust system to fully explore the power of siRNAs.
  
  
  OBJECTIVE
  We explored a siRNA expression cassette (SEC) to screen highly effective RNAi-targeted sequences for gene therapy of hepatocellular carcinoma (HCC).
  
  
  METHODS
  An SEC was developed by flanking H1 and U6 promoters in opposite directions at the siRNA-encoding sequence. Eight SECs specific to hTERT were designed by overlap extension polymerase chain reaction (PCR) and transfected into HepG2 cells with calcium phosphate. The telomerase activity was determined by telomeric repeat amplification protocol (TRAP) silver staining and TRAP real-time PCR analysis. The mRNA and protein expression levels of hTERT were determined by reverse transcription (RT)-PCR and western blot, respectively. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell apoptosis was measured by the annexin-V/propidium iodide (PI) assay coupled with flow cytometry.
  
  
  RESULTS
  Eight hTERT-specific SECs (SEC-1-8) were successfully constructed. In comparison to that of the negative control SEC, the hTERT-specific SECs, especially, SEC-4, SEC-5, SEC-7 and SEC-8 significantly reduced the activity of hTERT in HepG2 cells at 48 hours after transfection. Moreover, the mRNA and protein expression levels of hTERT as well as the cell viability were significantly reduced by SECs. Knockdown of hTERT by SECs in HepG2 cells led to cell apoptosis.
  
  
  CONCLUSIONS
  Our developed simple SEC was a powerful strategy for screening highly effective RNAi-targeted sequences and showed promise for gene therapy of HCC.
authors: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.
authors: Hui
authors: H
authors: Xu
authors: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.
authors: Xia
authors: X
authors: Gong
authors: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.
authors: Hui Hui
authors: HH
authors: Zhang
authors: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.
authors: Qin
authors: Q
authors: Zhang
authors: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.
authors: Dandan
authors: D
authors: Zhao
authors: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.
authors: Jian Xiong
authors: JX
authors: Peng
keywords: Gene Therapy; Hepatocellular Carcinoma; Telomerase; hTERT; siRNA