【摘要】 目的:HIF-1α是由低氧诱导表达的一个重要的调节肿瘤生长、代谢的转录因子,它的降解除了通过泛素-蛋白酶体途径降解之外还与可以通过细胞自噬途径降解。通过研究miR-147a对细胞自噬的影响从而进一步研究miR-147a对HIF-1α降解的影响。方法:在HeLa细胞中过表达miR-147a,用Western blot和Q-PCR检测细胞自噬相关的标志物LC3B、P62、LAMP-2A的变化。再通过溶酶体-自噬泡共定位实验共聚焦显微镜观察自噬泡的数量以及共定位情况。最后通过加入自噬诱导剂(EBSS)和自噬抑制剂(Bafilomycin A1),用Western blot检测转染NC与miR-147a后HIF-1α蛋白的表达情况。结果:过表达miR-147a后自噬相关的标志物LC3B、P62表达量上升,LAMP-2A表达量下降,且溶酶体与自噬泡的共定位增多;加入自噬诱导剂和自噬抑制剂后HIF-1α蛋白的表达量增加。结果表明miR-147a可以抑制细胞自噬的巨自噬途径以及分子伴侣介导的自噬途径,积累HIF-1α蛋白。结论:miR-147a通过抑制细胞自噬从而减少HIF-1α蛋白的降解,但是miR-147a作用靶点的分子机制需要进一步研究。更多还原

【Abstract】 Objective: HIF-1α is an important hypoxia-induced factor which regulates tumor growth and metabolism. And its degradation is associated with autophagy. To further study the impact of miR-147a on HIF-1α’degradation, we study the relationship between miR-147a and autophagy. Methods: MiR-147a was over-expressed in HeLa cells. Autophagy related markers, such as LC3 B,P62 and LAMP-2A were detected by Western blot and Q-PCR. Next, we used confocal microscope to observe the numbers of autophagic vacuoles and its co-localization with lysosome. Finally, we performed Western blot to detect the protein level of HIF-1α when HeLa cells were transfected with miR-147a and treated with autophagy inducer or autophagy inhibitor. Results: Autophagy related markers, such as LC3 B, P62 and LAMP-2A were increased and the numbers of autophagic vacuoles and its co-localization with lysosome were raised.The protein level of HIF-1α were increased when treated with autophagy inducer or autophagy inhibitor. All these experiments revealed that miR-147a could inhibit both macroautophagy and chaperone-mediated autophagy, thus reduced the degradation of HIF-1α.Conclusion: In this paper, we found that miR-147a could inhibit autophagy and accelerated the accumulation of HIF-1α through hindering the autophagy pathway of HIF-1α degradation. But the targets of miR-147a needed further research.更多还原