OBJECTIVE: To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression.
METHODS: Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation.
RESULTS: TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation.
CONCLUSIONS: The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway.
目的:阐明TQ在PC细胞中的作用及其对HIF-1α表达的调控机制。
方法:细胞计数试剂盒-8测定,进行Transwell分析和流式细胞术检测TQ对增殖活性的影响,PANC-1细胞和正常胰管上皮(hTERT-HPNE)细胞的迁移和侵袭能力以及凋亡。实时定量聚合酶链反应和免疫印迹法检测PC细胞中HIF-1αmRNA和蛋白的表达。通过Westernblot分析和免疫共沉淀检测TQ对PANC-1细胞中HIF-1α蛋白初始表达途径和泛素化降解的影响。
结果:TQ显著抑制增殖活性,迁移,和侵袭能力,促进PANC-1细胞凋亡;然而,未观察到对hTERT-HPNE细胞的显著影响。TQ显著降低PANC-1、AsPC-1和BxPC-3细胞中HIF-1α的mRNA和蛋白表达水平。TQ显著抑制HIF-1α初始表达通路(PI3K/AKT/mTOR)相关蛋白的表达,并促进PANC-1细胞中HIF-1α蛋白的泛素化降解。TQ对HIF-1α蛋白的羟基化和vonHippelLindau蛋白介导的泛素化降解没有影响,但通过抑制HIF-1α与HSP90之间的相互作用影响HIF-1α蛋白的稳定性,从而促进其泛素化降解。
结论:TQ对PC细胞HIF-1α蛋白表达的调控机制主要是通过抑制HIF-1α与HSP90的相互作用,促进HIF-1α蛋白的泛素化降解;其次,TQ通过抑制PI3K/AKT/mTOR途径降低HIF-1α蛋白的初始表达。