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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

Schematic Representation of the Overlap Extension PCR Amplification With DNA Pol III Promoters H1 and U6
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Related In: Results  -  Collection

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fig17913: Schematic Representation of the Overlap Extension PCR Amplification With DNA Pol III Promoters H1 and U6

Mentions: The plasmids pSUPER retro Neo + GFP with the H1 promoter and pRNAT-U6.1/Neo with the U6 promoter were from The Cancer Institute of Central South University (Changsha, China). The overlap extension PCR was designed to produce an SEC from the above two plasmids (Figure 1). These PCRs needed four primers. Two universal U6 forward and H1 forward primers were used in all PCR amplifications. The other two specific primers (U6 reverse primers and H1 reverse primers) were designed to contain the following sequences; a 19-nt siRNA targeting sequence, termination signal sequences of the other promoter and a promoter-specific sequence, which could anneal to the end of the template plasmid during PCR amplification.


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)

Schematic Representation of the Overlap Extension PCR Amplification With DNA Pol III Promoters H1 and U6
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig17913: Schematic Representation of the Overlap Extension PCR Amplification With DNA Pol III Promoters H1 and U6
Mentions: The plasmids pSUPER retro Neo + GFP with the H1 promoter and pRNAT-U6.1/Neo with the U6 promoter were from The Cancer Institute of Central South University (Changsha, China). The overlap extension PCR was designed to produce an SEC from the above two plasmids (Figure 1). These PCRs needed four primers. Two universal U6 forward and H1 forward primers were used in all PCR amplifications. The other two specific primers (U6 reverse primers and H1 reverse primers) were designed to contain the following sequences; a 19-nt siRNA targeting sequence, termination signal sequences of the other promoter and a promoter-specific sequence, which could anneal to the end of the template plasmid during PCR amplification.

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