Targeting Human Telomerase Reverse Transcriptase by a Simple siRNA Expression Cassette in HepG2 Cells

Area of Science:

• Molecular biology
• Cancer research
• Gene therapy

Background:

• Human telomerase reverse transcriptase (hTERT) is a key target for anticancer therapies.
• Small interfering RNAs (siRNAs) offer potent gene silencing but require effective screening methods.
• Developing robust systems to identify effective siRNAs against hTERT is crucial for cancer gene therapy.

Purpose of the Study:

• To explore a siRNA expression cassette (SEC) for screening highly effective RNAi-targeted sequences.
• To evaluate the potential of SECs for gene therapy of hepatocellular carcinoma (HCC).

Main Methods:

• An SEC was constructed using H1 and U6 promoters flanking an siRNA-encoding sequence.
• Eight hTERT-specific SECs were designed and transfected into HepG2 cells.
• Telomerase activity, hTERT expression (mRNA and protein), cell viability, and apoptosis were assessed.

Main Results:

• Eight hTERT-specific SECs were successfully created.
• Several SECs, particularly SEC-4, SEC-5, SEC-7, and SEC-8, significantly reduced hTERT activity in HepG2 cells.
• SEC-mediated hTERT knockdown led to decreased cell viability and induced apoptosis.

Conclusions:

• The developed SEC is a powerful strategy for screening effective RNAi sequences.
• This SEC system demonstrates significant promise for the gene therapy of HCC.