title: Construction and identification of multiple genes Co silence of plasmid shRNA.
abstract: Objective To construct and identify the eukaryotic vector expressing shRNA (Plasmid-1), which expressed the VEGF, C-myc, Survivin and hTERT gene at the same time. To detect its interference effects on the nasopharyngeal carcinoma cell line (CNE-2Z) compared with single gene plasmid VEFG (Plasmid-2). Methods According to the sequence of VEGF, C-myc, Survivin and hTERT gene, we designed 2 oligonucleotide sequences and synthesized a complementary DNA chain, then inserted it into the eukaryotic vector expressing pGenesil 1. The cell proliferation activity was detected by MTT method. The interference efficacy on human nasopharyngeal carcinoma cell line (CNE-2Z) in the level of mRNA and protein were detected by RT-PCR and Western-bolt. The inhibitory effect of plasmid on tumor in nude mice was also observed in vivo. Results The restriction enzyme digestion and sequencing technologies confirmed the construction of recombinant eukaryotic vector expressing was correct. The plasmid was transfected into CNE-2Z cells, green fluorescence can be seen clearly in the single gene and multi gene transfected cells under fluorescent microscope. MTT showed that the proliferation of cell was inhibited, the invasive ability was decreased in vitro, and the inhibition effects of single gene plasmid on the growth and proliferation of cells were lower than multi gene. Real-time-PCR and Western-bolt confirmed that the expression of target gene was decreased in the level of mRNA and protein, and the interference effect of multi gene was better than the single gene. The nude mice experiment showed that the interference effect of shRNA plasmid on the growth of tumor cell was better than single gene plasmid Conclusion We constructed a shRNA plasmid encoded four different genes successfully. After transfected with nasopharyngeal carcinoma cells, it can interfere the expression of VEGF, C-myc, Survivin and hTERT gene at the same time. And the interference effect was better than silence VEGF alone. Out results may provide experimental basis for multi gene therapy in the treatment of nasopharyngeal carcinoma.
authors.affiliation: Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University China.
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University China.
authors.forename: Liang
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University China.
authors.forename: Ming-Min
authors.initials: MM
authors.lastname: Dong
authors.affiliation: Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University China.
authors.forename: Hua
authors.initials: H
authors.lastname: Cao
authors.affiliation: Department of Otolaryngology, First Affiliated Hospital of Zhengzhou University China.
authors.forename: Xiu-Fen
authors.initials: XF
authors.lastname: Tian
keywords: Plasmid construction and identification; multi gene interference; nasopharyngeal carcinoma CNE-2Z cells; short hairpin RNA