Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (41): 7738-7741.doi: 10.3969/j.issn.1673-8225.2011.41.034

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Construction of survivin shRNA recombinant expression vector and downregulation of survivin mRNA expression in PC3 cells

Xu Jian-hua1, Chen Dan-na1, He Min1, Huang Xian-zhang1, Zhuang Jun-hua1, Li Mei-xian1, Jin Xiao-bao2, Lu Xue-mei2, Zhu Jia-yong2   

  1. 1Department of Clinical Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou  510120, Guangdong Province, China
    2Institute of Pharmaceutical Bioactive Substances of Guangdong University of Pharmacy, Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou  510006, Guangdong Province, China
  • Received:2011-04-20 Revised:2011-08-04 Online:2011-10-08 Published:2011-10-08
  • Contact: Zhu Jia-yong, Professor, Doctoral supervisor, Institute of Pharmaceutical Bioactive Substances of Guangdong University of Pharmacy, Guangdong Key Laboratory of Pharmaceutical Bioactive Substances, Guangzhou 510006, Guangdong Province, China zhujy@gdpu.edu.cn
  • About author:Xu Jian-hua☆, Studying for doctorate, Master’s supervisor, Associate chief technician, Department of Clinical Laboratory, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, Guangdong Province, China jhxu1976@126.com
  • Supported by:

    Guangdong Provincial Science and Technology Development Program, No. 2011B031800207*; a grant from Medical Science and Technology Research Program of Guangdong Province, No. A2008240*；Research Foundation for Young Talented Scholars of Guangdong Provincial Hospital of Chinese Medicine, No. E206001* 

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Abstract:

BACKGROUND: survivin mRNA is specifically expressed in tumor and embryonic tissue and is closely related to differentiation, proliferation, infiltration and metastasis of tumor cells as well as multidrug resistance.
OBJECTIVE: To construct the expression vector of small hairpin RNA (shRNA) targeting human survivin gene and detect the effectiveness of gene silencing in PC3 cells (human prostate cancer cell line).
METHODS: Two single-stranded DNA oligonucleotides for shRNA expression targeted survivin gene were chemically synthesized. The top and bottom strand oligos were annealed to generate a double-stranded oligonucleotide (ds oligo). The the oligo was cloned into pENTR/U6 expression vector (pENTR/U6-SUR), and then PCR and sequencing analyses were conducted to verify the constructs. After transfecting the verified plasmids into PC3 cells, RT-PCR was performed to determine the mRNA level of survivin gene.
RESULTS AND CONCLUSION: PCR and sequencing analyses demonstrated that shRNA template targeting survivin gene had been inserted at the expected site and the insertion sequence was perfectly corrected. The RT-PCR results showed that survivin expression in PC3 was downregulated at mRNA level. The recombinant plasmid had a better affect at 24 hours than 48 hours. The shRNA expression vector targeting survivin gene has been constructed successfully, and it would be a useful method to develop specific survivin-silencing therapeutics in further gene therapy study.

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