ABSTRACT
@#ObjectiveTo observe the growth of BT-325 human glioma cells after interfering volume-regulated chloride channel ClC-2 gene.MethodsTwo expression recombinant vectors of ClC-2 gene were designed and constructed. The primary plasmid, pSUPER.puro-shRNA, and the two recombinant plasmids, pSUPER.puro-shRNA-ClC-21 and pSUPER.puro-shRNA-ClC-22, were transfected into BT-325 cells by LipofectamineTM2000 (Groups: control, PP1 and PP2, respectively). The mRNA expression of ClC-2 gene was detected with reverse transcription polymerasse chain reaction (RT-PCR), the cellular survival was determined with MTT assay, and the cell cycle was measured with flow cytometry (FCM). ResultsClC-2 mRNA expression and the growth of the cells in PP1 and PP2 groups were significantly lower than that of control group. The cell cycle progression was blocked in G1 phase (PP1 and PP2 vs control,P<0.01). ConclusionThe growth of BT-325 human glioma cells is prevented by knockdown of ClC-2 gene expression, which may be one of the novel targets to inhibit growth of human malignant glioma cells.
ABSTRACT
Objective :To construct the small interfering RNA(siRNA) eukaryotic expression vectors of human ClC-2 gene. Methods: According to the program and principles of DEQOR about designing siRNA,two pairs ClC-2 mRNA-targeted hairpin siRNA were devised,and the two pairs complemantary oligonucleotide strands of DNA fragments that encoded the above siRNA were synthesized by chemosynthesis.After annealing of the complementary strands,the DNA fragments were connected to the polyclone sites of plasmid eukaryotic expression vector pSUPER.puro that was cut by restriction endonuclease BglⅡ and HindⅢ,followed by transformation,amplification and plasmid extraction in E.coli,and finally,the two recombinant plasmids were identified by agarose gel electrophoresis by means of cutting with EcoRⅠand HindⅢ and by DNA sequence analysis.The plasmids were transfected transiently into human glioma cell line BT-325 cells by Lipofectamine~(TM)2000.The mRNA expression of ClC-2 gene was detected by(RT-PCR.) Results: The connections between the DNA fragments encoding ClC-2-targeted siRNA and the pSUPER.puro plasmid were correct,as confirmed by agarose gel electrophoresis and DNA sequencing(analysis.) ClC-2 mRNA expression of the BT-325 cells transfected two recombinant vectors was(significantly) decreased.Conclusion: The two RNAi recombinant vectors of human ClC-2 gene were successfully constructed.They were named pSUPER.puro-siClC-21 and pSUPER.puro-siClC-22,respectively.This laid the groundwork for future research about ClC-2 gene affecting invasion and migration of human glioma cells.