Establishment of Human Neuroblastoma SH-SY5Y Cell Line Stably Silencing Beclin1_Journal of Biomedical Engineering

Authors：

WEIChuanjie , XIAOShuang , JIANGLan , TANYan , HUANGBo ,  LONGDingxin

School of Public Health, University of South China, Hengyang 421001 Hunan, China;

Corresponding author：

LONGDingxin, Email: dxlong99@163.com

Keywords：

JM109; SH-SY5Y; pGenesil-1-Beclin1; G418 screening; cell cloning

DOI：

10.7507/1001-5515.20140204

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The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E.coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28%±1.45%(P<0.05)and 75.50%±2.63%(P<0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SY5Y cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.

Citation： WEIChuanjie, XIAOShuang, JIANGLan, TANYan, HUANGBo, LONGDingxin. Establishment of Human Neuroblastoma SH-SY5Y Cell Line Stably Silencing Beclin1. Journal of Biomedical Engineering, 2014, 31(5): 1085-1089. doi: 10.7507/1001-5515.20140204 Copy

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1. HUANG J, KLIONSKY D J. Autophagy and human disease[J]. Cell Cycle, 2007, 6(15): 1837-1849.
2. MCCORMICK J, KNIGHT R A, BARRY S P, et al. Autophagy in the stress-induced myocardium[J]. Front Biosci (Elite Ed), 2012, 4:2131-2141.
3. KONDO Y, KONDO S. Autophagy and cancer therapy[J]. Autophagy, 2006, 2(2): 85-90.
4. SHINTANI T, KLIONSKY D J. Autophagy in health and disease: a double-edged sword[J]. Science, 2004, 306(5698): 990-995.
5. KONGARA S, KARANTZA V. The interplay between autophagy and ROS in tumorigenesis[J]. Front Oncol, 2012, 2:171.
6. ZHAO L, ZHU Y, WANG D, et al. Morphine induces Beclin 1-and ATG5-dependent autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus[J]. Autophagy, 2010, 6(3): 386-394.
7. YUE Z, HORTON A, BRAVIN M, et al. A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice[J]. Neuron, 2002, 35(5): 921-933.
8. LIANG X H, JACKSON S, SEAMAN M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1[J]. Nature, 1999, 402(6762): 672-676.
9. AITA V M, LIANG X H, MURTY V V, et al. Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21[J]. Genomics, 1999, 59(1): 59-65.

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