miRNA-146通过调控TLR4/NF-κB信号通路促进 POI模型卵巢颗粒细胞生长的机制研究

Mechanistic study of miRNA-146 promoting the growth of ovarian granulosa cells in a POI model by regulating the TLR4/NF-κB signaling pathway

目的 探讨小鼠原发性卵巢功能不全(POI)模型的微小RNA(miRNA)-146、Toll样受体4(TLR4)和核转录因子-κB(NF-κB)的水平及对卵巢颗粒细胞生长的影响。方法 取健康雌性BALB/c小鼠40只,随机分为POI组和对照组,每组20只。采用实时荧光定量PCR(qRT-PCR)检测血清miRNA-146水平;采用酶联免疫吸附试验(ELISA)检测肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)表达水平;采用蛋白质印迹法检测小鼠卵巢组织TLR4和NF-κB蛋白水平。采用miRNA-146模拟物或抑制剂转染小鼠卵巢颗粒细胞,评估卵巢颗粒细胞增殖、生长活力和细胞凋亡率及TLR4、NF-κB通路蛋白的表达。采用Spearman相关分析POI模型miRNA-146水平与TLR4和NF-κB水平的相关性。结果 POI组的miRNA-146水平显著低于对照组(P<0.05),TNF-α、IL-6、TLR4、NF-κB水平高于对照组(P<0.05)。miRNA-146模拟物在卵巢颗粒细胞中呈高表达水平,且呈浓度依赖趋势。miRNA-146转染后,miRNA-146模拟物组细胞凋亡率低于对照组,miRNA-146抑制剂组细胞凋亡率高于对照组,差异均有统计学意义(P<0.01)。Spearman相关性分析结果显示,POI小鼠的miRNA-146水平与TLR4、NF-κB水平呈负相关(r=-0.725、-0.681,P<0.05)。结论 上调miRNA-146水平具有促进卵巢颗粒细胞生长的作用,其机制可能与miRNA-146负性调控TLR4/NF-κB信号通路和下调TNF-α、IL-6的表达相关,miRNA-146有望作为评估卵泡质量的指标之一。

Objective To investigate the expression levels of microRNA(miRNA)-146,Toll-like receptor 4(TLR4),and nuclear transcription factor-κB(NF-κB)and their effects on ovarian granulosa cell growth in a mouse model of primary ovarian insufficiency(POI).Methods Forty healthy female BALB/c mice were taken and randomly divided into POI group and control group,with 20 mice in each group.The expression of miRNA-146 in serum was detected by qRT-PCR.The expression levels of inflammatory factors tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by ELISA.The expression levels of TLR4 and NF-κB protein in mouse ovarian tissue were detected by Western blotting.Mouse ovarian granulosa cells were transfected with miRNA-146 mimics or inhibitors,and ovarian granulosa cells were evaluated for proliferation,growth viability,and apoptosis rates as well as expression of TLR4 and NF-κB pathway proteins.Correlation of miRNA-146 levels in POI model with TLR4 and NF-κB levels was analyzed by Spearman correlation analysis.Results Compared with the control group,the expression level of miRNA-146 in POI group decreased significantly(P<0.05),the expression levels of TNF-α,IL-6,TLR4,NF-κB increased significantly(P<0.05).miRNA-146 mimics were expressed at high levels in ovarian granulosa cells in a concentration-dependent manner.After miRNA-146 transfection,the apoptosis rate of miRNA-146 mimics group was lower than that of the control group,and the apoptosis rate of miRNA-146 inhibitor group was higher than that of the control group,and the differences were all statistically significant(P<0.01).Spearman correlation analysis showed that miRNA-146 levels in POI mice were negatively correlated with TLR4 levels and NF-κB levels(r=-0.725,-0.681,P<0.05).Conclusion Up-regulation of miRNA-146 expression level has the effect of promoting the growth of ovarian granulosa cells,and its mechanism may be related to the negative regulation of the TLR4/NF-κB signaling pathway and down-regulation of the expression of TNF-αand IL-6 by miRNA-146,which is expected to be used as one of the indexes for assessing follicle quality.