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Cell-specific expression of the analgesic-antitumor peptide coding sequence under the control of the human α-fetoprotein gene promoter and enhancer.

Jin S, Lin X, Guan H, Wu J - Exp Ther Med (2015)

Bottom Line: The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed.The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP.Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, P.R. China.

ABSTRACT

The aim of the present study was to construct a gene-modified hepatocellular carcinoma (HCC)-specific analgesic-antitumor peptide (AGAP) expression vector regulated by the α-fetoprotein (AFP) promoter and enhancer, in order to evaluate its effect. The AFP promoter is generally used in HCC-specific gene therapy strategies. However, this approach is limited by the weak activity of the AFP promoter. Linking the AFP enhancer and promoter has been shown to generate a stronger and more HCC-selective promoter. The AGAP DNA fragment was amplified from the total RNA of the Chinese scorpion, Buthus martensii Karsch. The fragment was subsequently cloned into the pAFP plasmid with the minimal essential DNA fragment, which included the AFP gene promoter and enhancer, to construct the recombinant plasmid, pAFP-AGAP. The plasmid was transfected into HepG2 cells and the mRNA expression levels of AGAP were detected by reverse transcription polymerase chain reaction (RT-PCR). In addition, Cell Counting Kit 8 (CCK-8) was used to analyze the cytotoxicity of plasmid transfection. The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed. Using RT-PCR and CCK-8 analysis, the mRNA expression levels of AGAP and the cytotoxicity in AFP-producing human HCC cells were determined. The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP. Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.

No MeSH data available.


Related in: MedlinePlus

HepG2 cells were transfected with the pAFP-AGAP plasmid and harvested at (A) 24, (B) 48 and (C) 72 h. AFP, α-fetoprotein; AGAP, analgesic-antitumor peptide.
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f2-etm-09-03-0863: HepG2 cells were transfected with the pAFP-AGAP plasmid and harvested at (A) 24, (B) 48 and (C) 72 h. AFP, α-fetoprotein; AGAP, analgesic-antitumor peptide.

Mentions: HepG2 cells were transfected with the pAFP-AGAP complex. The cells grew normally after 24 h (Fig. 2A); however, cellular swelling and cell suspension was observed 48 h after cultivation (Fig. 2B). The presence of floating cells, cytoplasmic granulations and vacuolation was observed after 72 h (Fig. 2C). This phenomenon indicated that the cytotoxicity increased gradually over time. The AGAP gene was amplified using RT-PCR from the RNA of the cells simultaneously. The products following RT-PCR of the HepG2 cells transfected with the pAFP-AGAP plasmid for 24, 48 and 72 h were confirmed to be the correct size by gel electrophoresis. However, the same product was not found on the electrophoresis gel following RT-PCR of the HepG2 cells transfected with the pAFP plasmid only (control; Fig. 3).


Cell-specific expression of the analgesic-antitumor peptide coding sequence under the control of the human α-fetoprotein gene promoter and enhancer.

Jin S, Lin X, Guan H, Wu J - Exp Ther Med (2015)

HepG2 cells were transfected with the pAFP-AGAP plasmid and harvested at (A) 24, (B) 48 and (C) 72 h. AFP, α-fetoprotein; AGAP, analgesic-antitumor peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4316983&req=5

f2-etm-09-03-0863: HepG2 cells were transfected with the pAFP-AGAP plasmid and harvested at (A) 24, (B) 48 and (C) 72 h. AFP, α-fetoprotein; AGAP, analgesic-antitumor peptide.
Mentions: HepG2 cells were transfected with the pAFP-AGAP complex. The cells grew normally after 24 h (Fig. 2A); however, cellular swelling and cell suspension was observed 48 h after cultivation (Fig. 2B). The presence of floating cells, cytoplasmic granulations and vacuolation was observed after 72 h (Fig. 2C). This phenomenon indicated that the cytotoxicity increased gradually over time. The AGAP gene was amplified using RT-PCR from the RNA of the cells simultaneously. The products following RT-PCR of the HepG2 cells transfected with the pAFP-AGAP plasmid for 24, 48 and 72 h were confirmed to be the correct size by gel electrophoresis. However, the same product was not found on the electrophoresis gel following RT-PCR of the HepG2 cells transfected with the pAFP plasmid only (control; Fig. 3).

Bottom Line: The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed.The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP.Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, P.R. China.

ABSTRACT

The aim of the present study was to construct a gene-modified hepatocellular carcinoma (HCC)-specific analgesic-antitumor peptide (AGAP) expression vector regulated by the α-fetoprotein (AFP) promoter and enhancer, in order to evaluate its effect. The AFP promoter is generally used in HCC-specific gene therapy strategies. However, this approach is limited by the weak activity of the AFP promoter. Linking the AFP enhancer and promoter has been shown to generate a stronger and more HCC-selective promoter. The AGAP DNA fragment was amplified from the total RNA of the Chinese scorpion, Buthus martensii Karsch. The fragment was subsequently cloned into the pAFP plasmid with the minimal essential DNA fragment, which included the AFP gene promoter and enhancer, to construct the recombinant plasmid, pAFP-AGAP. The plasmid was transfected into HepG2 cells and the mRNA expression levels of AGAP were detected by reverse transcription polymerase chain reaction (RT-PCR). In addition, Cell Counting Kit 8 (CCK-8) was used to analyze the cytotoxicity of plasmid transfection. The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed. Using RT-PCR and CCK-8 analysis, the mRNA expression levels of AGAP and the cytotoxicity in AFP-producing human HCC cells were determined. The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP. Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.

No MeSH data available.


Related in: MedlinePlus