miR-130b-3p靶定AMPK基因抑制糖饥饿诱导的宫颈癌细胞自噬性死亡

miR-130b-3p inhibits autophagic cell death induced by glucose starvation in cervical cells by targeting AMPK gene

目的探究miR-130b-3p对糖饥饿诱导宫颈癌细胞自噬性死亡的影响及机制。方法将宫颈癌细胞分别置于低糖(0.1 mmol/L)和正常糖含量(5 mmol/L)、低糖+DMSO和低糖+雷帕霉素的培养液中培养,利用碘化丙啶(propidium iodide,PI)染色实验、GFP-LC3B自噬泡形成检测实验、实时荧光半定量PCR(RT-qPCR)实验和Western blot实验,依次检测细胞死亡、自噬水平,细胞内miR-130b-3p、腺苷单磷酸激活激酶(AMP-activated protein kinase,AMPK)mRNA和AMPK蛋白水平;分别转染miR-130b-3p抑制物及AMPK过表达质粒,再利用自噬抑制剂SBI-0206965(SBI)检测miR-130b-3p、ampk对细胞死亡和自噬的影响;生物信息学工具预测miR-130b-3p的靶基因并利用荧光报告基因实验进行验证。结果低糖诱导宫颈癌细胞发生自噬、miR-130b-3p水平升高,AMPK表达水平降低(均P<0.05),死亡率无显著变化(均P>0.05);与低糖+DMSO组相比,低糖+雷帕霉素使得宫颈癌细胞死亡率显著增加(均P<0.05);转染miR-130b-3p抑制物后,低糖条件下宫颈癌细胞自噬水平和自噬性死亡率显著增加(均P<0.05);过表达AMPK能促进宫颈癌细胞自噬和自噬性死亡率显著升高(均P<0.05);荧光报告基因表达水平检测结果证实,AMPK是miR-130b-3p的直接靶基因(P<0.05)。结论低糖条件下,miR-130b-3p靶定AMPK基因抑制宫颈癌细胞自噬性死亡。

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 rapamycin-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-LC3 B assay,semiquantitative 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 inhibitor 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 confirmed that AMPK was the direct target gene of miR-130b-3p(P<0.05).Conclusion Under low glucose conditions,miR-130b-3p inhibits autophagic cell death in cervical cancer cells by targeting the AMPK gene.