Key Lab for Critical Care Medicine of the Ministry of Health, Affiliated Tianjin First Center Hospital, Tianjin Medical University, Tianjin, 300192, China.
J Exp Clin Cancer Res. 2010 Jan 14;29(1):3. doi: 10.1186/1756-9966-29-3.
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.
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.
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.
To our knowledge, this is the first description of a role for connexin 43 in the inhibition of U251 growth using Ad-bFGF-siRNA.
碱性成纤维细胞生长因子（bFGF）是促进神经胶质瘤细胞增殖和侵袭的重要生长因子，而连接蛋白 43 则参与了神经胶质瘤生长的抑制。相应地，神经胶质瘤的连接蛋白 43 表达已经被证明减少或受损。
在这项研究中，使用腺病毒（Ad-bFGF-siRNA）将 bFGF 靶向 siRNA 递送至神经胶质瘤细胞系 U251，并评估连接蛋白 43 的表达及其磷酸化状态。将 U251 细胞用 Ad-bFGF-siRNA（100、50 或 25 MOI）感染，用表达绿色荧光蛋白的腺病毒（Ad-GFP）感染 100 MOI 作为对照。用 Western 印迹和免疫荧光检测感染和未感染 Ad-bFGF-siRNA 的 U251 细胞中连接蛋白 43 的表达水平、磷酸化和定位。
与感染 Ad-GFP 的细胞相比，用 100 和 50 MOI 的 Ad-bFGF-siRNA 感染的 U251 细胞中连接蛋白 43 的水平明显升高，而在感染 Ad-GFP 的和未感染的 U251 细胞中检测到相同量的连接蛋白 43。感染 Ad-bFGF-siRNA 的 U251 细胞中的连接蛋白 43 磷酸化没有差异。然而，Ad-bFGF-siRNA 细胞中磷酸化与非磷酸化连接蛋白 43 的比值较低，连接蛋白 43 主要定位于细胞质。使用刮伤加载染料转移测定法，与对照组相比，用 Ad-bFGF-siRNA 处理的组中更多的 Lucifer Yellow 转移到相邻细胞中。
据我们所知，这是首次描述使用 Ad-bFGF-siRNA 时连接蛋白 43 在抑制 U251 生长中的作用。
