DFG-STYK1/NOK对小鼠胚胎成纤维细胞系NIH3T3增殖以及细胞周期G_(1)/S的影响

Influence of DFG-STYK1/NOK on cell proliferation and G_(1)/S distribution in mouse fibroblast cell line NIH3T3

目的研究新型受体酪氨酸激酶STYK1/NOK加入DFG基序后对小鼠胚胎成纤维细胞系NIH3T3增殖和细胞周期G_(1)/S的影响。方法SWISS MODEL同源比对STYK1/NOK的二级结构确定DFG加入的位置;用CCK8和MTT法检测瞬时转染pcDNA3.0或pcDNA3.0-DFG-NOK的NIH3T3细胞增殖;流式细胞测量术检测细胞周期;Western blot检测细胞增殖信号通路蛋白Akt、p-Akt、Erk、p-Erk、STAT、p-STAT、JAK3、p-JAK3和周期蛋白cyclin D1的表达。结果与瞬时转染pcDNA3.0相比,1)瞬时转染pcDNA3.0-DFG-NOK可抑制NIH3T3细胞增殖;2)DFG-NOK阻碍细胞周期由G_(1)期进入S期;3)DFG-NOK抑制Erk、STAT1、STAT3和JAK3的磷酸化和周期蛋白cyclin D1表达。结论DFG-NOK抑制NIH3T3细胞增殖和细胞周期G_(1)期进入S期。

Objective To investigate the effects of NOK added DFG motif on proliferation and G_(1)/S distribution of NIH3T3 cells.Methods SWISS MODEL homologous modeling was used to confirm the location of DFG addition;we used CCK-8 and MTT methods to measure the cell proliferation of NIH3T3 transiently transfected pcDNA3.0 or pcDNA3.0-DFG-NOK;Flow cytometry was used to measure cell cycle;Western-blot was used to exam the phosphorylation of Akt,Erk,STAT1,STAT3,JAK3 and the expression of cyclin D1.Results Compared with the pcDNA3.0 transient transfection:1)The proliferation of pcDNA3.0-DFG-NOK over-expression NIH3T3 cells was inhibited.2)The entrance of G_(1)/S in NIH3T3 cells transiently transfected pcDNA3.0-DFG-NOK was obstructed.3)Transient transfection of pcDNA3.0-DFG-NOK could inhibit phosphorylation of Erk,STAT1,STAT3,JAK3 and expression of CyclinD1.Conclusions DFG-NOK inhibits proliferation of NIH3T3 cells and obstructs cell cycle G_(1) phase entering into S phase.