Construction of lentiviral vector for RNA interference of Id2 gene and its effect on proliferation and apoptosis of gliomas cell line in vitro
10.3724/SP.J.1008.2011.00598
- Author:
Zhen-Yu ZHAO
1
Author Information
1. Department of Neurosurgery
- Publication Type:Journal Article
- Keywords:
Apoptosis;
Cell proliferation;
Glioma;
Inhibitor of differentiation protein 2;
RNA interference
- From:
Academic Journal of Second Military Medical University
2011;32(6):598-602
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a lentiviral vector carrying short hairpin RNA (shRNA) of human Id2 gene and to investigate its effect on the proliferation and apoptosis in glioma cell line U251. Methods Four shRNA sequences targeting human Id2 gene were designed and synthesized. The shRNAs were inserted into the pGCSIL-GFP lentiviral vector. Id2 sequence was amplified from the plasmid which contained Id2 gene as verified by PCR, and was inserted into the pEGFP-N1-3FLAG vector. Lentiviral vector for RNAi of Id2 was constructed and was used to infect 293T cells. The infection efficiency was evaluated and then the lentiviral vector was used to infect U251 cellline. Western blotting analysis was employed to assess the gene silencing efficiency. Cell proliferation was tested by MTT. Cell apoptosis was observed by RT-PCR analysis of caspase 3 expression in U251 cells after infection. Results The results of PCR analysis and DNA sequencing agreed to each other. Compared with the control group, the transfected U251 cells had significantly decreased cell proliferation, and increased apoptosis rate and caspase 3 expression (P<0.05).Conclusion We have successfully constructed lentivirus RNAi vector of Id2, which can inhibit the proliferation and promote the apoptosis of glioma cells in vitro.
