Down-regulation of tankyrase 1 mediated by RNAi leads to the apoptosis of SH-SY5Y cells

doi:10.3969/j.issn.0529-1356.2014.02.010

Abstract

Abstract:

Objective To investigate the effect and mechanism of tankyrase 1（TNKS1）down-regulation mediated by RNA interference（RNAi）on the proliferation and apoptosis of human neuroblastoma (NB) SH-SY5Y cells, and to provide experimental basis for the development of a new therapeutical target. Methods According to TNKS1 gene sequences, three short hairpin (shRNA) interference segments with gene-specific were designed and synthesized, and lentiviral vectors were used for constructing RNAi sequence. After SH-SY5Y cells were transfected with lentiviral vector, Real-time PCR assay was used for detecting the expression of TNKS1 gene, and the best interference sequence was screened out for subsequent experiments. The colony forming assay was used to detect the change of cell proliferation after RNAi-TNKS1. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein Caspase-3, the key protein β-catenin of Wnt/β-catenin pathway and its downstream target proteins Cyclin D1 and c-Myc were detected by the Western blotting method for exploring the mechanism. Apoptotic morphology was observed by transmission electron microscopy. Results The lentiviral vector was constructed successfully, and the virus concentration was 5×10 11TU/L. The results of Real-time PCR test showed that #3 shRNA was the most effective target sequence. The colony forming assay results showed that colony forming rate decreased significantly after RNAi-TNKS1 compared with that of control group. The results of Western blotting showed that the protein expression of Bcl-2 reduced significantly as well as β-catenin, Cyclin D1 and c-Myc after RNAi-TNKS1, while the protein expression of caspase-3 increased. The transmission electron microscopy results showed significant apoptotic structure and morphology after RNAi-TNKS1. Conclusion The down-regulation of TNKS1 mediated by RNAi leads to the proliferation decrease and the apoptosis of SH-SY5Y cells, and the inhibition of Wnt/β-catenin pathway may play a role in it.

TIAN Xiao-hong BAI Shu-ling* FAN Jun HOU Wei-jian TONG Hao XU He. Down-regulation of tankyrase 1 mediated by RNAi leads to the apoptosis of SH-SY5Y cells[J]. AAS, 2014, (2): 196-203.

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