敲降WDR1抑制STAT3的核转移调控平滑肌细胞的增殖和迁移

Knockdown of WDR1 Inhibits Nuclear Translocation of STAT3 to Regulate Smooth Muscle Cell Proliferation and Migration

信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)的核转运是其发挥生物学功能的前提。WD重复结构域1(WD repeat domain 1,WDR1)作为细胞骨架的调节因子可能影响STAT3的核转运。然而,有关WDR1影响STAT3核转运的分子机制仍不清楚。平滑肌细胞的增殖和迁移是动脉狭窄的主要原因。为了探究在平滑肌细胞中WDR1对STAT3核转运的影响,本研究构建了WDR1稳定敲降的人主动脉血管平滑肌细胞(human aortic vascular smooth muscle cells,HAVSMCs)模型。RT-qPCR和Western印迹结果显示,敲降WDR1,STAT3的活化和表达未见明显改变(P>0.05);核质分离结果表明,与对照组相比,敲降WDR1后STAT3的核质分布受到显著影响。随后的研究结果表明,与对照组相比,STAT3核转运相关的核输入蛋白β(importinβ)表达受到抑制(P<0.05),Ras相关核蛋白(Ras-related nuclear protein,Ran)的核质比显著下降。CCK8和Transwell结果表明,过表达importinβ能够挽救由WDR1敲降引起的HAVSMCs增殖和迁移的抑制。进一步研究结果表明,敲降WDR1导致与Ran核苷酸循环有关的核转运相关因子2(nuclear transport factor 2,NTF2)的表达显著下降(P<0.05)。过表达NTF2后,CCK8和Transwell结果表明,HAVSMCs的增殖和迁移能力得到显著提升(P<0.05)。总结上述结果可见,敲降WDR1通过抑制核输入蛋白β和核转运相关因子2的表达,改变Ran的核质分布,降低STAT3的核转运,进而调控平滑肌细胞的增殖和迁移。

Nuclear transport of signal transducer and activator of transcription 3(STAT3)is a prerequisite for its biological function.WD Repeat domain 1(WDR1),a regulator of the cytoskeleton factors,may affect STAT3 nuclear translocation.However,the molecular mechanism regarding the effect of WDR1 on STAT3 nuclear translocation is still unknown.To investigate the effect of WDR1 on STAT3 nuclear translocation in smooth muscle cells,a human aortic vascular smooth muscle cells(HAVSMCs)model with stable knockdown of WDR1 was constructed.The results of RT-qPCR and Western blot showed that the activation and expression of STAT3 were not significantly altered after knockdown of Wdr1(P>0.05);the results of nucleoplasm isolation showed that the nucleoplasm distribution of STAT3 was significantly affected after knockdown of WDR1 compared with the control group.Subsequent results showed that the expression of STAT3 nuclear input-associated proteinβ(importinβ)was inhibited(P<0.05)and the nucleoplasmic ratio of Ras-associated nuclear protein(Ran)was significantly decreased compared to the control group.Results from CCK8 and Transwell assays indicated that overexpression of importinβwas able to rescue the inhibition of proliferation and migration of HAVSMCs caused by WDR1 knockdown.Further results showed that knockdown of WDR1 resulted in a significant decrease in the expression of nuclear transport factor 2(NTF2)associated with the Ran nucleotide cycle(P<0.05).After overexpression of NTF2,the results of CCK8 and Transwell experiments showed that the proliferation and migration ability of HAVSMCs were significantly enhanced(P<0.05).Summarizing the above results,knockdown of WDR1,by inhibiting the expression of importinβand NTF2,alters the nucleoplasmic distribution of Ran and decreases the nuclear translocation of STAT3,thus regulating the proliferation and migration of smooth muscle cells.