Adenovirus-mediated delivery of bFGF small interfering RNA increases levels of connexin 43 in the glioma cell line, U251.
BACKGROUND
bFGF is an important growth factor for glioma cell proliferation and invasion, while connexin 43 is implicated in the suppression of glioma growth. Correspondingly, gliomas have been shown to have reduced, or compromised, connexin 43 expression.


METHODS
In this study, a bFGF-targeted siRNA was delivered to the glioma cell line, U251, using adenovirus (Ad-bFGF-siRNA) and the expression of connexin 43 and its phosphorylation state were evaluated. U251 cells were infected with Ad-bFGF-siRNA (100, 50, or 25 MOI), and infection with adenovirus expressing green fluorescent protein (Ad-GFP) at 100 MOI served as a control. Western blotting and immunofluorescence were used to detect the expression levels, phosphorylation, and localization of connexin 43 in U251 cells infected, and not infected, with Ad-bFGF-siRNA.


RESULTS
Significantly higher levels of connexin 43 were detected in U251 cells infected with Ad-bFGF-siRNA at 100 and 50 MOI than in cells infected with Ad-GFP, and the same amount of connexin 43 was detected in Ad-GFP-infected and uninfected U251 cells. Connexin 43 phosphorylation did not differ between Ad-bFGF-siRNA-infected and uninfected U251 cells. However, the ratio of phosphorylated to unphosphorylated connexin 43 in Ad-bFGF-siRNA cells was lower, and connexin 43 was predominantly localized to the cytoplasm. Using a scrape loading dye transfer assay, more Lucifer Yellow was transferred to neighboring cells in the Ad-bFGF-siRNA treated group than in the control group.


CONCLUSIONS
To our knowledge, this is the first description of a role for connexin 43 in the inhibition of U251 growth using Ad-bFGF-siRNA.
Zhang
Biao
B
Key Lab for Critical Care Medicine of the Ministry of Health, Affiliated Tianjin First Center Hospital, Tianjin Medical University, Tianjin, 300192, China.
Feng
Xuequan
X
Wang
Jinhuan
J
Xu
Xinnu
X
Liu
Hongsheng
H
Lin
Na
N
D000256:Adenoviridae / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism; D002450:Cell Communication; D045744:Cell Line, Tumor; D018031:Connexin 43 / Q000378:metabolism*; D015536:Down-Regulation; D005346:Fibroblast Growth Factors / Q000235:genetics*; D005822:Genetic Vectors; D005910:Glioma / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics*
D018031:Connexin 43; D034741:RNA, Small Interfering; C108111:fibroblast growth factor 13; D005346:Fibroblast Growth Factors