Construction of RNAi recombinant expression vectors of targeting wnt-1 gene and study of its growth inhibition effect on glioma cell U251

Objective To construct the RNAi expression vectors of wnt-1 and transfected glioma cell line U251 to study its effect by this plasmid.Methods According to the sequence of the coding region of wnt-1 gene,two strings of 21 nucleotides of inverted sequence flanking the loop sequence of two complementary 21-base oligonucleotides were designed and synthesized to form hairpin construction as the DNA templates for the target siRNA.The siRNA templates were cloned into siRNA expression vector pGPU6/GFP/Neo,and the sequence of the plasmid was identified by DNA sequencer and restriction endonuclease digestion.Thus,the resulted vector pGPU6/GFP/Neo-shRNA-wnt-1 was transfected into U251 cells.RT-PCR and Western blotting were performed to evaluate wnt-1 gene silencing induced by siRNA transfection at mRNA and the protein levels.Preliminary study of its growth effect on glioma cell U251 was measured by MTT and FCM.Results It was verified that the specific DNA oligonucleotide was cloned into the vector successfully,and the protein expression of wnt-1 gene in U251 cells was significantly reduced after transfecting the recombinant plasmid,compared to the controls.Western blotting revealed significantly lowered wnt-1 expression at protein levels in transfected U251 cells,which exhibited a significantly higher death rate after transfection as shown by MTT.Conclusion It indicates the shRNA expression vector has been successfully established which can inhibit the protein expression of wnt-1 gene,and that provides the precondition for the further study of wnt-1 by wnt signaling pathway in glioma pathogenesis.