TARGETING HUMAN TELOMERASE REVERSE TRANSCRIPTASE BY A SIMPLE SIRNA EXPRESSION CASSETTE IN HEPG2 CELLS

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XU HUI; GONG XIA; ZHANG HUI HUI; ZHANG QIN; ZHAO DANDAN; PENG JIAN XIONG

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Journal Paper

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Journal: Hepatitis Monthly Year:2015 | Volume:15 | Issue:3 Page(s): 0-0

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Title

TARGETING HUMAN TELOMERASE REVERSE TRANSCRIPTASE BY A SIMPLE SIRNA EXPRESSION CASSETTE IN HEPG2 CELLS

Author(s)

Keywords

HTERT

SIRNA

TELOMERASE

HEPATOCELLULAR CARCINOMA

GENE THERAPY

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.Objectives: We explored a SIRNA expression cassette (SEC) to screen highly effective RNAi-targeted sequences for GENE THERAPY of HEPATOCELLULAR CARCINOMA (HCC).Materials and 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.

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References

EVALUATION OF THE PATHOGENESIS OF PSEUDOMONAS AERUGINOSA'S FLAGELLUM BEFORE AND AFTER FLAGELLAR GENE KNOCKDOWN BY SMALL INTERFERING RNAS (SIRNA)

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APA: Copy

XU, HUI, GONG, XIA, ZHANG, HUI HUI, ZHANG, QIN, ZHAO, DANDAN, & PENG, JIAN XIONG. (2015). TARGETING HUMAN TELOMERASE REVERSE TRANSCRIPTASE BY A SIMPLE SIRNA EXPRESSION CASSETTE IN HEPG2 CELLS. HEPATITIS MONTHLY, 15(3), 0-0. SID. https://sid.ir/paper/306900/en

Vancouver: Copy

XU HUI, GONG XIA, ZHANG HUI HUI, ZHANG QIN, ZHAO DANDAN, PENG JIAN XIONG. TARGETING HUMAN TELOMERASE REVERSE TRANSCRIPTASE BY A SIMPLE SIRNA EXPRESSION CASSETTE IN HEPG2 CELLS. HEPATITIS MONTHLY[Internet]. 2015;15(3):0-0. Available from: https://sid.ir/paper/306900/en

IEEE: Copy

HUI XU, XIA GONG, HUI HUI ZHANG, QIN ZHANG, DANDAN ZHAO, and JIAN XIONG PENG, “TARGETING HUMAN TELOMERASE REVERSE TRANSCRIPTASE BY A SIMPLE SIRNA EXPRESSION CASSETTE IN HEPG2 CELLS,” HEPATITIS MONTHLY, vol. 15, no. 3, pp. 0–0, 2015, [Online]. Available: https://sid.ir/paper/306900/en

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