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

Xu H, Gong X, Zhang HH, Zhang Q, Zhao D, Peng JX - Hepat Mon (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.

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.

No MeSH data available.


Related in: MedlinePlus

hTERT-Specific SECs Reduced the Proliferation of HepG2 CellsHepG2 cells were transfected with the indicated SECs or controls for 1-5 days. Cell viability was determined by the MTT assay. Mock, calcium phosphate reagent only; NC, negative control; 1, SEC-4; 2, SEC-7.
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fig17918: hTERT-Specific SECs Reduced the Proliferation of HepG2 CellsHepG2 cells were transfected with the indicated SECs or controls for 1-5 days. Cell viability was determined by the MTT assay. Mock, calcium phosphate reagent only; NC, negative control; 1, SEC-4; 2, SEC-7.

Mentions: To assess the effect of SECs on cell proliferation, we treated HepG2 cells with SEC-4 and SEC-7 and examined cell proliferation by the MTT assay. As shown in Figure 6, from the third day, the proliferation rates in the SEC-4 and SEC-7 groups were significantly lower than the control group (P < 0.05).


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

Xu H, Gong X, Zhang HH, Zhang Q, Zhao D, Peng JX - Hepat Mon (2015)

hTERT-Specific SECs Reduced the Proliferation of HepG2 CellsHepG2 cells were transfected with the indicated SECs or controls for 1-5 days. Cell viability was determined by the MTT assay. Mock, calcium phosphate reagent only; NC, negative control; 1, SEC-4; 2, SEC-7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385270&req=5

fig17918: hTERT-Specific SECs Reduced the Proliferation of HepG2 CellsHepG2 cells were transfected with the indicated SECs or controls for 1-5 days. Cell viability was determined by the MTT assay. Mock, calcium phosphate reagent only; NC, negative control; 1, SEC-4; 2, SEC-7.
Mentions: To assess the effect of SECs on cell proliferation, we treated HepG2 cells with SEC-4 and SEC-7 and examined cell proliferation by the MTT assay. As shown in Figure 6, from the third day, the proliferation rates in the SEC-4 and SEC-7 groups were significantly lower than the control group (P < 0.05).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory, Xiangya Medial School, Central South University (CSU), Changsha, China.

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.

No MeSH data available.


Related in: MedlinePlus