关键词:

miR?130b?3p, AMPK, 自噬性细胞死亡, 宫颈癌

Abstract:

Objective To explore the effect and mechanism of miR⁃130b⁃3p on glucose starvation induced autophagic cell death in cervical cancer cells. Methods Cervical cancer cells were cultivated in DMSO or rapamy⁃ cin⁃containing low level of glucose（0.1 mmol/L）and standard glucose（5 mmol/L）media，low level of glucose + DMSO and low level of glucose + rapamycin，respectively. Propidium iodide staining assay，GFP⁃LC3B assay，semi⁃ quantitative real⁃time PCR（RT⁃qPCR）and Western blot assays were subsequently employed to determine the rate of cell death，autophagy level and the expression levels of miR⁃130b⁃3p，AMPK mRNA and AMPK protein in cervical cancer cells. The effects of miR⁃130b⁃3p and ampk on autophagy and cell death ratio were determined by transfecting miR⁃130b⁃3p inhibitor and the plasmid overexpressing the AMPK gene，then，after stimulation with autophagy inhib⁃ itor SBI⁃0206965，respectively. Multiple bioinformatics techniques were used to predict miR⁃130b⁃3p target genes， which were then confirmed using a fluorescence reporter gene assay. Results Low glucose induced autophagy， increased miR⁃130b⁃3p levels，decreased AMPK expression levels（P < 0.05），and no significant changes in mortality in cervical cancer cells（P > 0.05）；low level of glucose + rapamycin dramatically boosted autophagy and death in cervical cancer cells when compared to the low level of glucose + DMSO group. After transfection of miR⁃130b⁃3p inhibitor，cervical cancer cell autophagy levels and autophagic cell death ratio increased significantly under low glucose conditions（P < 0.05）；overexpression of AMPK can promote autophagy and autophagic cell death ratio of cervical cancer cells significantly increased（P < 0.05）. Fluorescence reporter gene expression level detection results con⁃ firmed that AMPK was the direct target gene of miR⁃130b⁃3p（P < 0.05）. Conclusion Under low glucose condi⁃ tions，miR⁃130b⁃3p inhibits autophagic cell death in cervical cancer cells by targeting the AMPK gene.

Key words:

miR?130b?3p, AMPK, autophagic cell death, cervical cancer