JNK信号通路对NaAsO2诱导NIH3T3细胞中hnRNP K蛋白聚集体形成的影响

 目的：观察核不均一核糖核蛋白(hnRNP)K的表达与定位以及亚砷酸钠（NaAsO2）刺激NIH3T3细胞的反应情况及氧化应激变化,探讨JNK信号通路在hnRNP K蛋白聚集体形成中的作用。方法：构建重组载体pcDNA3-hnRNP K-HA,转染NIH3T3细胞,荧光显微镜观察内源性hnRNP K在细胞中的表达与定位,以及在NaAsO2不同刺激时间该蛋白聚集体的形成情况,并利用活性氧（ROS）检测试剂盒测定胞内ROS水平;给予JNK、MEK、PI3K/Akt、NF-κB和核转运等5种信号通路的抑制剂预处理后,观察聚集体的变化情况。结果：重组质粒构建正确,荧光显微镜观察显示hnRNP K主要定位于胞核中,而重组质粒转染NIH3T3细胞后,可见胞内有蛋白聚集体形成并随NaAsO2刺激时间的延长而递增,且过表达hnRNP K可抑制NaAsO2刺激细胞生成ROS;JNK信号通路抑制剂SP600125明显抑制聚集体的形成。结论：hnRNP K主要定位在NIH3T3细胞的胞核中,胞浆少量分布;NaAsO2可诱导NIH3T3细胞形成hnRNP K蛋白聚集体,此聚集体可抑制胞内ROS生成,且该聚集体的形成有赖于JNK介导的信号通路。

Effects of JNK signaling pathway on hnRNP K protein aggregates in NIH3T3 cells treated with sodium arsenite

AIM: To explore the role of JNK signaling pathway in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein aggregates by observing the expression and localization of hnRNP K in NIH3T3 cells induced by sodium arsenite(NaAsO2).METHODS: The recombinant vector pcDNA3-hnRNP K-HA was constructed and transfected into NIH3T3 cells. The expression and localization of endogenous hnRNP K and distribution of the protein aggregates stimulated with NaAsO2 at different time points were observed under fluorescence microscope. The level of reactive oxygen species (ROS) in the cells was detected by a ROS assay kit. After pretreated with the inhibitors of 5 signaling pathways (JNK, MEK, PI3K/Akt, NF-κB and nuclear transport), the changes of the protein aggregates were observed.RESULTS: The recombinant plasmid was correctly constructed. The hnRNP K was mainly distributed in the nucleus. The intracellular fluorescent aggregates increased with the prolonging stimulation of NaAsO2 in the cells transfected with the recombinant plasmid, and the overexpression of hnRNP K inhibited ROS generation in the cells induced by NaAsO2. SP600125, an inhibitor of JNK signaling pathway, significantly inhibited the formation of protein aggregates. CONCLUSION: The hnRNP K is primarily located in the nucleus of NIH3T3 cells, with a small amount in the cytoplasm. The formation of protein aggregates is significant after NaAsO2 stimulation, which can restrain the level of intracellular ROS, and the process is involved in JNK signaling pathway.