雷帕霉素靶蛋白复合物1对Hela细胞微管蛋白稳定性的影响

目的: 探讨雷帕霉素靶蛋白复合物1 (mammalian target of rapamycin complex 1,mTORC1）对Hela细胞微管稳定性的影响及可能机制。方法: 使用50 nmol/L雷帕霉素预处理Hela细胞24 h后，加入5 μmol的诺考达唑处理30、60和120 min，免疫荧光检测微管蛋白的稳定性；使用50 nmol/L雷帕霉素处理Hela细胞24 h后，蛋白质印迹检测促微管解聚蛋白stathmin、KIF2A，促微管聚合蛋白CLIP170，微管切割蛋白Katanin、Spastin的表达变化。转染ATG5-shRNA抑制自噬相关基因5（autophagy-related gene5， ATG5）后，免疫荧光检测微管蛋白稳定性的改变。使用50 nmol/L雷帕霉素预处理Hela细胞24 h后，蛋白质印迹检测Ras同源基因家族成员A(Ras homolog gene family member A,RhoA)的表达；转染GFP RhoA-Q63L或GFP RhoA-N19、p190RhoGAP-siRNA质粒后，免疫荧光检测微管稳定性的改变。结果： 用雷帕霉素抑制mTORC1的活性后Hela细胞微管的稳定性增强，但微管稳定性相关蛋白stathmin、KIF2A、CLIP170、Katanin、Spastin无明显变化。抑制细胞自噬后，微管的稳定性无明显改变。雷帕霉素抑制 mTORC1的活性后RhoA GTP酶的活化水平下调；下调p190RhoGAP的活化水平后，微管的稳定性减弱。结论： RhoA在mTORC1介导的Hela细胞微管稳定性中起重要作用。

Effect of mTORC1 on the stability of microtubule in Hela cells#br#

Objective: To investigate the effect and mechanism of mammalian target of rapamycin complex 1（mTORC1）on the stability of microtubules in Hela cells. Methods: Hela cells was pretreated with rapamycin（50 nmol/L）for 24 hours, then treated with Nocodazole（5 μmol）for 30, 60 or 120 minutes, the stability of microtubule protein was detected by immunofluorescence. Hela cells was treated with rapamycin(50 nmol/L) for 24 hours, the expression of microtubule depolymerizing protein stathmin, KIF2A, microtubule polymerizing protein CLIP170, microtubule cutting protein Katanin and Spastin were detected by Western blotting. The stability of microtubule protein was detected by immunofluorescence after autophagy related gene 5 (ATG5) was inhibited by ATG5-shRNA. Hela cells was pretreated with rapamycin(50 nmol/L) for 24 hours, the expression of Ras homologous gene family member A (RhoA) was detected by Western blotting; the stability of microtubules was examined by immunofluorescence after transfection with GFP RhoA-Q63l or GFP RhoA-N19, P190RhoGAP-siRNA plasmids. Results: Inhibiting the activity of mTORC1 with rapamycin significantly enhanced the stability of microtubules through resisting depolymerization while the stathmin, KIF2A, CLIP170, Katanin and Spastin expression were not changed.The stability of microtubules mediated by mTORC1 was independent on the autophagy. After the activity of mTORC1 was inhibited by rapamycin, the activation of RhoA GTP was down-regulated and the stability of microtubule was attenuated along with decreased activation of P190 RhoGAP. Conclusion: mTORC1 regulated the stability of microtubules through  RhoA pathway in Hela cells. ［Key words］mTORC1; microtubules; RhoA; autophagy； Hela cells