Abstract
A Wingless-type MMTV integration site family, member 1 (Wnt-1) RNA interference expression vector was constructed during the present study, which was used to transfect the glioma U251 cell line and investigate its effect on glioma. Two 21-base oligonucleotides complementary to the coding sequence that was flanking the loop sequence were designed to form a DNA hairpin template for the target small interfering RNA (siRNA). The siRNA templates were cloned into the siRNA expression vector, pGPU6/green fluorescent protein (GFP)/Neo and the sequence was confirmed by DNA sequencing. The pGPU6/GFP/Neo-short hairpin RNA (shRNA)-Wnt-1 vector was subsequently transfected into U251 cells, and reverse transcription polymerase chain reaction and western blot analysis were used to evaluate the Wnt-1 gene silencing effect on U251 cell growth by MTT assay and flow cytometry. The Wnt-1 protein expression was significantly reduced following transfection with the recombinant plasmid, as determined by western blot analysis of the transfected U251 cells. This transfection exhibited a significantly higher death rate, as shown by MTT. Thus, the present study demonstrated that the pGPU6/GFP/Neo-shRNA-Wnt-1 vector inhibited Wnt-1 protein expression. However, further investigations regarding the Wnt signaling pathway in glioma pathogenesis are required.
Highlights
Wingless‐type MMTV integration site, member 1 (Wnt‐1), the first identified member of the Int‐1 family, was named by Nusse in 1991 [1]
Preliminary experiments in the present study found that the Wnt‐1 protein expression in human glioma was significantly higher than in normal brain tissue, and is closely associated with the degree of malignancy
The vectors used were as follows: pGPU6/green fluorescent protein (GFP)/Neo‐short hairpin RNA‐GAPDH served as a positive control and pGPU6/GFP/Neo‐shNC served as a negative control
Summary
Wingless‐type MMTV integration site, member 1 (Wnt‐1), the first identified member of the Int‐1 family, was named by Nusse in 1991 [1]. The first Int‐1 was termed Wnt‐1, as Int‐1 and the Wnt gene family were homologous. Current studies have proposed that the Wnt‐1 protein controls cell growth, proliferation, secretion of key signaling molecules, mediates information flow between cells and stem cells, and is important in neural development. Wnt‐1 gene expression has been found to closely correlate with the development of numerous types of tumor [1,2,3,4,5,6,7]. Preliminary experiments in the present study found that the Wnt‐1 protein expression in human glioma was significantly higher than in normal brain tissue, and is closely associated with the degree of malignancy. The aim of the current study was to further investigate the role of Wnt‐1 in glioma by RNA interference (RNAi) targeting
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