微核糖核酸-125a通过调控细胞凋亡和炎症反应延缓阿尔茨海默病进展的研究

microRNA-125a attenuates Alzheimer’s disease progression via regulating neuron apoptosis and inflammation

目的 探究微核糖核酸-125a(mircoRNA-125a,简称miR-125a)在PC12阿尔茨海默病(Alzheimer’s disease, AD)细胞模型中,对细胞凋亡、总神经突生长和炎症因子水平的影响。方法 采用Aβ1-42毒化神经生长因子诱导的PC12细胞株构建PC12 AD模型细胞,采用荧光定量聚合酶链式反应检测miR-125a相对表达量。将miR-125a模拟物(mimic)、对照mimic、miR-125a抑制剂(inhibitor)和对照inhibitor转染至PC12 AD模型细胞后,统计Hoechst 33342阳性细胞数和碘化丙啶阳性细胞数,计算细胞凋亡率;于显微镜下观察细胞形态,计算总神经突生长长度;采用ELISA法测定细胞上清液中炎症因子水平。结果 Aβ1-42组miR-125a相对表达量显著少于对照组(P<0.05)。miR-125a mimic组细胞凋亡率为(7.55±1.56)%,显著低于对照mimic组的(13.00±1.11)%(P<0.05);miR-125a inhibitor组细胞凋亡率为(19.34±2.41)%,显著高于对照inhibitor组的(13.11±2.04)%(P<0.01)。miR-125a mimic组中乳酸脱氢酶(LDH)毒性细胞比例为(6.14±1.20)%,显著低于对照mimic组的(15.43±2.55)%(P<0.05);miR-125a inhibitor组中LDH毒性细胞比例为(23.07±3.86)%,显著高于对照inhibitor的(14.79±2.26)%(P<0.05)。miR-125a mimic组总神经突生长长度为(34.54±2.95)μm,较对照mimic组的(23.93±2.64)μm显著增长(P<0.01)。miR-125a inhibitor组总神经突生长长度为(18.47±2.27)μm,较对照inhibitor组的(24.62±1.52)μm显著缩短(P<0.05)。miR-125a mimic组中TNF-α、IL-1β、IL-6、IL-17水平较对照mimic组均显著降低(P<0.01或0.001)。miR-125a inhibitor组中TNF-α、IL-1β、IL-6、IL-17水平较对照inhibitor组均显著增高(P<0.05或0.01)。miR-125a mimic组中脑源性神经营养因子(BDNF) mRNA和蛋白质相对表达量均较对照mimic组显著增加(P值均<0.01)。miR-125a inhibitor组中BDNF mRNA和蛋白质相对表达量均相较对照inhibitor组显著减少(P值均<0.01)。结论 miR-125a可能对Aβ1-42毒化的分化PC12细胞株具有保护作用。

Objective To investigate the effect of microRNA-125a(miR-125a) on cell apoptosis, neurite outgrowth and inflammation level in cell models of PC12 Alzheimer’s disease(AD). Methods PC12 AD cell models were established via treating nerve growth factor(NGF) stimulated PC12 cells with Aβ1-42. The expression of miR-125a was detected via quantitative polymerase chain reaction assay. Then miR-125a mimic, blank mimic, miR-125a inhibitor and blank inhibitor plasmids were transfected into PC12 AD cells. Cell apoptosis was detected via Hoechst/propidium iodide assay. Total length of neurite outgrowth of the cells was observed. Inflammatory factor levels were detected by enzyme-linked immunosorbent assay(ELISA). Results The relative expression of miRNA-125a in PC12 AD cell models was significantly lower than that in controls(P<0.05). The cell apoptosis rate in miR-125a mimic group was significantly lower than that in blank mimic group([7.55±1.56]% vs. [13.00±1.11]%, P<0.05), but the cell apoptosis rate in miR-125a inhibitor group was significantly higher than that in blank inhibitor group([19.34±2.41]% vs. [13.11±2.04]%, P<0.01). The proportion of lactate dehydrogenase(LDH) cytotoxicityin miR-125a mimic group was significantly lower than that in blank mimic group([6.14±1.20]% vs. [15.43±2.55]%, P<0.05), but the proportion in miR-125a inhibitor group was significantly higher than that in blank inhibitor group([23.07±3.86]% vs. [14.79±2.26]%, P<0.05).Total neurite outgrowth in miR-125a mimic group was significantly longer than that in blank mimic group([34.54±2.95] μm vs. [23.93±2.64] μm, P<0.01), while the total neurite outgrowth was significantly decreased in miR-125a inhibitor group compared with blank inhibitor group([18.47±2.27] μm vs. [24.62±1.52] μm, P<0.05). The levels of tumor necrosis factor(TNF)-α, interleukin(IL)-1β, IL-6 and IL-17 in miR-125a mimic group were significantly lower than those in blank mimic group(all P<0.01, 0.001), while the levels of TNF-α, IL-1β, IL-6 and IL-17 in miR-125a inhibitor group were significantly higher than those in blank inhibitor group(P<0.05, <0.01). The relative expressions of brain-derived neurotrophic factor(BDNF) mRNA and protein in miR-125a mimic group were significantly higher than those in blank mimic group(both P<0.01), but the relative expressions of BDNF mRNA and protein in miR-125a inhibitor group were significantly lower than those in blank inhibitor group(both P<0.01). Conclusion MiR-125a may have a protective effect on Aβ1-42 poisoned differentiated PC12 cells.