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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。
OBJECTIVE: HIF-1α is an important hypoxia-inducible transcription factor that regulates tumor growth and metabolism. Its degradation is not only through the ubiquitin-proteasome pathway but also through autophagy pathway. The effects of miR-147a on the degradation of HIF-1αwere further investigated by studying the effect of miR-147a on autophagy. Methods: miR-147a was overexpressed in HeLa cells and the changes of autophagy-related markers LC3B, P62 and LAMP-2A were detected by Western blot and Q-PCR. The number of autophagic vacuoles and co-localization were observed by confocal microscopy with lysosome-autophagy co-localization experiment. Finally, the expression of HIF-1α protein in NC and miR-147a cells was detected by Western blot after adding EBSS and Bafilomycin A1. Results: The expression of autophagy markers LC3B, P62 increased, the expression of LAMP-2A decreased, and the co-localization of lysosomes and autophagic vacuoles increased after miR-147a was overexpressed. The addition of autophagy inducer and autophagy inhibition After the agent HIF-1α protein expression increased. The results indicate that miR-147a can accumulate HIF-1α protein by inhibiting the autophagy of the macrophage pathway and chaperone-mediated autophagy pathway. Conclusion: miR-147a can reduce the degradation of HIF-1α protein by inhibiting autophagy. However, the molecular mechanism of miR-147a needs further study.