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Impact of Co-transfection with Livin and Survivin shRNA Expression Vectors on Biological Behavior of HepG2 Cells

  • Xu, Wei (Department of General Surgery, Provincial Hospital Affiliated to Shandong University) ;
  • Chang, Hong (Department of General Surgery, Provincial Hospital Affiliated to Shandong University) ;
  • Qin, Cheng-Kun (Department of General Surgery, Provincial Hospital Affiliated to Shandong University) ;
  • Zhai, Yun-Peng (Department of General Surgery, Provincial Hospital Affiliated to Shandong University)
  • Published : 2013.09.30

Abstract

Objective: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting Livin and Survivin genes, and to explore the impact of co-transfection of Livin and Survivin shRNA expression vectors on the biological behavior of HepG2 cells. Methods: shRNA eukaryotic expression vectors pSD11-Livin and pSD11-Survivin were designed and constructed then transfected into HepG2 cells separately or in combination. mRNA and protein expression in transfected cells was assessed by quantitative fluorescence PCR and Western blotting, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by TUNEL assay. Results: The Livin and Survivin shRNA eukaryotic expression vectors were successfully constructed and transfected into HepG2 cells. The relative mRNA expression levels of Livin and Survivin in HepG2 cells co-transfected with pSD11-Livin and pSD11-Survivin were $0.12{\pm}0.02$ and $0.33{\pm}0.13$, respectively, which was significantly lower than levels in cells transfected with either pSD11-Livin or pSD11-Survivin (P<0.05). The relative protein expression levels of Livin and Survivin in the co-transfected cells were also significantly decreased compared to single-transfection (P<0.05). The inhibition rate of cell growth in the co-transfection group was higher than that in the single-transfection groups at 48 h, 60 h, or 72 h after transfection (P<0.01). The apoptotic rate increased to the greatest extent in the co-transfection group relative to any other group (P<0.05). Conclusions: Co-transfection with pSD11-Livin and pSD11-Survivin was more efficient than transfection with either vector alone in reducing the mRNA and protein expression of Livin and Survivin genes in HepG2 cells. Co-transfection also inhibited the proliferation of transfected cells more than the other groups, and induced cellular apoptosis more effectively.

Keywords

Livin;Survivin;RNA interference;hepatocellular carcinoma

Acknowledgement

Supported by : Natural Science Foundation

References

  1. Apasani K (2007). RNA interference technology-from basic science to drug development. 125-35.
  2. Augello C, Caruso L, Maggioni M, et al (2009). Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma. BMC Cancer, 9, 125. https://doi.org/10.1186/1471-2407-9-125
  3. Brummelkamp TR, Bernards R, Agami R (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550-3. https://doi.org/10.1126/science.1068999
  4. Carrasco RA, Stamm NB, Marcusson E, et al (2011). Antisense inhibition of survivin expression as a cancer therapeutic. Mol Cancer Ther, 10, 221-32. https://doi.org/10.1158/1535-7163.MCT-10-0756
  5. Crnkovic-Mertens I, Hoppe-Seyler F, Butz K (2003). Induction of apoptosis in tumor cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene. Oncogene, 22, 8330-6. https://doi.org/10.1038/sj.onc.1206973
  6. Chang H, Li S, Su Z (2008). Expression of Livin, Survivin, and IGF-2 in hepatocellular carcinoma tissue and its clinical implications. Shandong Med, 48, 1-3.
  7. Liu H, Wang S, Sun H, et al (2010). Inhibition of tumorigenesis and invasion of hepatocellular carcinoma by siRNAmediated silencing of the livin gene. Mol Med Rep, 3, 903-7.
  8. Hung CS, Liu HH, Huang MT, et al (2012). Knockdown survivin expression reduces the efficacy of curcumin treatment in hepatocellular carcinoma cells. Ann Surg Oncol, 19, 3547-55 https://doi.org/10.1245/s10434-012-2393-4
  9. Kenneth NS, Duckett CS (2012). IAP proteins: regulators of cell migration and development. Curr Opin Cell Biol, 24, 871-5. https://doi.org/10.1016/j.ceb.2012.11.004
  10. Lau R, Pratt MA (2012). The opposing roles of cellular inhibitor of apoptosis proteins in cancer. ISRN Oncol, 2012, 928120.
  11. Liu X, Wang A, Gao H, et al (2012). Expression and role of the inhibitor of apoptosis protein livin in chemotherapy sensitivity of ovarian carcinoma. Int J Oncol, 41, 1021-8. https://doi.org/10.3892/ijo.2012.1540
  12. Oh BY, Lee RA, Kim KH (2011). siRNA targeting Livin decreases tumor in a xenograft model for colon cancer. World J Gastroenterol, 17, 2563-71. https://doi.org/10.3748/wjg.v17.i20.2563
  13. Portt L, Norman G, Clapp C, et al (2011). Anti-apoptosis and cell survival: a review. Biochim Biophys Acta, 1813, 238-59. https://doi.org/10.1016/j.bbamcr.2010.10.010
  14. Sibley CR, Seow Y, Wood MJ (2010). Novel RNA-based strategies for therapeutic gene silencing. Mol Ther, 18, 466-76. https://doi.org/10.1038/mt.2009.306
  15. Song J, Cao L, Li Y (2013). RNA interferencemediated inhibition of survivin and VEGF in pancreatic cancer cells in vitro. Mol Med Rep, 7, 1651-5. https://doi.org/10.3892/mmr.2013.1361
  16. Song J, Giang A, Lu Y, et al (2008). Multiple shRNA expressing vector enhances efficiency of gene silencing. BMB Rep, 41, 358-62. https://doi.org/10.5483/BMBRep.2008.41.5.358
  17. Wang R, Lin F, Wang X, et al (2008). Silencing Livin gene expression to inhibit proliferation and enhance chemosensitivity in tumor cells. Cancer Gene Ther, 15, 402-12. https://doi.org/10.1038/cgt.2008.16
  18. Xing J, Jia CR, Wang Y, et al (2012). Effect of shRNA targeting survivin on ovarian cancer. J Cancer Res Clin Oncol, 138, 1221-9. https://doi.org/10.1007/s00432-012-1196-0
  19. Yang D, Song X, Zhang J, et al (2010). Therapeutic potential of siRNA-mediated combined knockdown of the IAP genes (Livin, XIAP, and Survivin) on human bladder cancer T24 cells. Acta Biochim Biophys Sin (Shanghai), 42, 137-44. https://doi.org/10.1093/abbs/gmp118

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