shRNA干扰S100A13基因对人甲状腺癌细胞释放成纤维细胞生长因子1的影响

目的 探讨S100A13基因沉默对应激诱导人甲状腺癌细胞(TT细胞)成纤维细胞生长因子1(FGF-1)释放的影响.方法 将S100A13-shRNA pENTRTM/U6入门质粒转染TT细胞,应用实时PCR、间接免疫荧光法及Western印迹方法检测细胞内S100A13基因及蛋白表达的抑制效率,应用间接免疫荧光,RT-PCR及ELISA检测无血清处理TT细胞S100A13沉默后FGF-1释放变化.结果 S100A13-shRNApENTRTM/U6入门载体转染TT细胞后能够抑制S100A13基因及蛋白表达,抑制效率约为80%.间接免疫荧光法显示FGF-1主要位于细胞浆和细胞核,无血清培养6 h后胞浆内FGF-1基本消失.RT-PCR和ELISA结果均显示,S100A13沉默能降低无血清处理TT细胞培养上清中FGF-1的浓度(P＜0.05).结论 S100A13-shRNA pENTRTM/U6入门载体能有效、特异性地抑制S100A13基因及蛋白表达.S100A13基因的抑制可以减弱FGF-1从细胞内释放到细胞外的过程.

Effects of small hairpin RNA-mediated S100A13 gene inhibition on the release of fibroblast growth factor-1 in human thyroid cancer cells

Objective To investigate whether the release of fibroblast growth factor-1 ( FGF-1 ) was changed after inhibition of S100A13 gene (small hairpin RNA, shRNA)and serum-deprivation in human thyroid cancer cells (TT cells ). Methods The S100A13-shRNA pENTRTM/U6 entry vector was transfected into TT cells. The expression of S100A13 mRNA and protein was detected by immunoflurescence, real-time RT-PCR, and Western blot. Then TT cells were treated with S100A13 gene inhibition and serum-deprivation. The changes in release of FGF-1 were detected by indirect immunoflurescence, RT-PCR, and ELISA. Results S100A13 shRNA transfected TT cells (S100A13 RNAi cells)had a reduction of S100A13 gene and protein expression by 80%.Indirect immunofluorescence indicated FGF-1 was mostly localized in the cytoplasm and nucleus of TT cells in primary culture. When serum-deprivation stress was given to TT cells, FGF-1 in cytoplasm almost disappeared in the cells at 6 h. RT-PCR indicated that when serum-deprivation stress was given to TT cells the mRNA of FGF-1 was reduced. ELISA showed that with inhibition of S100A13, the release of FGF-1 was reduced (P＜0.05).Conclusion S100A13-shRNA pENTRTM/U6 entry vector transfected TT cells may inhibit the expression of S100A13 and reduce the release of FGF-1.