miR-34a-5p靶向AKT1基因对子宫内膜异位症子宫内膜基质细胞侵袭及自噬的调控

microRNA-34a-5p regulates invasion and autophagy of endometrial stromal cells in endometriosis by inhibiting AKT1

目的研究微小RNA-34a-5p(miR-34a-5p)靶向AKT1基因对子宫内膜异位症(EM)子宫内膜基质细胞(ESCs)侵袭及自噬的调控作用。方法原代分离、培养EM患者ESCs,构建negative control RNA(NC)及mimic细胞模型,采用双荧光肾素酶报告系统验证miR-34a-5p与AKT1基因存在结合位点,RT-PCR及Western blotting检测并验证2组细胞miR-34a-5p和AKT1基因的表达及调控关系;检测miR-34a-5p对ESCs的增殖、迁移、侵袭、凋亡的影响。结果EM患者ESCs中miR-34a-5p较非EM患者明显降低,而AKT1基因的表达明显升高,且两者变化呈负相关;miR-34a-5p通过特异性结合AKT1基因的3’UTR区对AKT1基因的表达进行调控;转染miR-34a-5p mimic可使ESCs中miR-34a-5p表达升高,AKT1基因表达降低,同时使ESCs的增殖、迁移及侵袭能力增强,而凋亡及自噬能力下降。结论miR-34a-5p靶向AKT1基因促进ESCs的增殖、迁移及侵袭能力,降低其凋亡及自噬能力,可能在EM的发病过程中发挥作用。

Objective To investigate the effect of microRNA-34a-5p(miR-34a-5p)on the invasion and autophagy of endometrial stromal cells(ESCs)during endometriosis(EM).Methods ESCs were isolated from EM patients and the expression of miR-34a-5p and AKT1 in ESCs were analyzed by RT-PCR and the expression of AKT1 protein was detected by Western blotting.A dual-luciferase reporter assay was used to dissect the interaction between miR-34a-5p and AKT1.The proliferation,migration,invasion,and apoptosis of ESCs transfected with miR-34a-5p mimic or negative control(NC)was measured with the CCK-8 assay,wound-healing assay,Transwellinvasion assay,and flow cytometry,respectively.Results During EM,the expression of miR-34a-5p in the ESCs was significantly lower than that in non-EM patients(P<0.05),while AKT1 expression was significantly higher.miR-34a-5p could specifically bind the 3’-UTR of AKT1 and regulate its expression.The expression of AKT1 in the ESCs transfected with the miR-34a-5p mimic was lower than in the NC group.Thus,overexpression of miR-34a-5p can reduce the proliferation,migration,and invasion of ESCs in EM and enforce apoptosis.Conclusion miR-34a-5p promotes the viability of ESCs by targeting AKT1,which may play a crucial role in the pathogenesis of EM.