微小RNA-29a/PTEN通路在铝致神经元网络损伤中的作用及机制
Role and mechanism of miRNA-29a/PTEN pathway in neuronal network damage caused by aluminum
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摘要:
背景 铝可致海马突触可塑性损伤，其机制可能是神经元间信号传递受阻。微小RNA-29a（miR-29a）能够靶向调控第10号染色体同源丢失性磷酸酶张力蛋白基因（PTEN）的表达，参与神经元网络的生成，并可能参与铝对神经元网络电活动的影响。
目的 通过体外培养经过麦芽酚铝Al（mal）3处理的ICR小鼠原代海马神经元，研究miR-29a靶向调控PTEN在铝对神经元网络损伤中的作用及机制。
方法 体外培养24 h内新生的ICR乳鼠原代海马神经元，在培养第6天时使用免疫荧光化学法标记神经元特异性蛋白微管相关蛋白2（MAP2）确定神经元的纯度；使用不同浓度的Al（mal）3处理神经元，将神经元分为对照组，10、20、40 μmol·L−1 Al（mal）3组，采用CCK-8法检测神经元细胞活力，后续实验选择20 μmol·L−1 Al（mal）3建立神经元网络损伤模型进行干预。采用慢病毒感染的方法转染miR-29a进入神经元，分为mNG组、mNG+20 μmol·L−1 Al（mal）3组、miR-29a组、miR-29a+20 μmol·L−1 Al（mal）3组，使用微电极阵列（MEA）分析神经元及神经网络的放电情况，使用实时定量PCR（RT-PCR）检测各组miR-29a和PTEN mRNA的表达情况，采用Western blotting检测各组神经元PTEN蛋白的表达情况。
结果 小鼠原代海马神经元的纯度大于90%，各组原代海马神经元细胞活力均在80%以上。在Al（mal）3处理48 h时，接种于MEA板上的对照组神经元自发放电频率、簇发放电频率、网络簇发放电频率和同步指数变化幅度分别为207.56%±38.70%、73.19%±46.43%、75.42%±33.04%和117.13%±15.54%，Al（mal）3处理组神经元网络电活动呈现下降的趋势。与对照组相比，20、40 μmol·L−1 Al（mal）3组的自发放电频率、簇发放电频率、网络簇发放电频率和同步指数明显下降（变化幅度分别为171.70%±28.08%、49.20%±23.23%、50.20%±18.18%、85.45%±20.30%和150.68%±26.15%、43.43%±15.54%、52.05%±26.31%、26.80%±8.29%，均P < 0.05）。与对照组（1.00）相比，20、40 μmol·L −1 Al（mal）3组miR-29a的相对表达水平分别下降为0.74±0.09和0.62±0.12（均P < 0.05）， PTEN mRNA的相对表达水平分别升高为1.32±0.12和1.48±0.11（均P < 0.05），PTEN蛋白相对表达量分别升高到1.29±0.12和1.82±0.10（均 P < 0.05）。使小鼠原代海马神经元过表达miR-29a，与mNG组相比，miR-29a组的自发放电频率、簇发放电频率和网络簇发放电频率明显升高（变化幅度分别为252.80%±62.03%、171.65%±56.30%和197.75%±27.12%，均 P < 0.05），mNG+20 μmol·L −1 Al（mal）3组的神经元网络电活动明显下降（各参数变化幅度分别为123.28%±47.31%、66.62%±31.53%、70.60%±12.48%和52.86%±20.26%，均P < 0.05）。与mNG+20 μmol·L −1 Al（mal）3组相比，miR-29a+20 μmol·L−1 Al（mal）3组的神经元网络电活动明显升高（各参数变化幅度分别为161.41%±42.13%、101.16%±30.63%、127.02%±29.58%和109.73%±15.61%，均P < 0.05）。与mNG组（1.00）相比，miR-29a组神经元 PTEN mRNA相对表达量（0.67±0.11）明显下降（P < 0.05），PTEN蛋白表达（0.75±0.08）下降（ P < 0.05）；mNG+20 μmol·L −1 Al（mal）3组PTEN mRNA相对表达量（1.32±0.12）明显升高（P < 0.05），PTEN蛋白相对表达量（1.46±0.15）上升（ P < 0.05）。与mNG+20 μmol·L −1 Al（mal）3组比较，miR-29a+20 μmol·L−1 Al（mal）3组PTEN mRNA相对表达量（0.93±0.06）降低（P < 0.05），PTEN蛋白相对表达量（0.92±0.09）降低（ P < 0.05）。
结论 铝显著抑制海马神经元网络电活动，miR-29a通过调控PTEN的表达可能参与铝致海马神经元网络电活动损伤。
Abstract:Background Aluminum can cause synaptic plasticity damage in the hippocampus, probably due to blocked interneuronal signal transmission. MicroRNA-29a (miR-29a) can target phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and participate in the generation of neuronal networks, and may be involved in the effect of aluminum on the electrical activity of neuronal networks.
Objective To study the role and mechanism of miR-29a-targeted PTEN in aluminum-induced neuronal network injury in primary hippocampal neurons of ICR mice treated with maltol aluminum Al(mal)3 in vitro.
Methods Primary hippocampal neurons of ICR mice born within 24 h were cultured in vitro. The purity of neurons was determined by labeling neuron-specific microtubule-associated protein 2 (MAP2) by immunofluorescence staining on day six of the culture; neurons were treated with different concentrations of Al(mal)3, and divided into a control group, and 10, 20, and 40 μmol·L−1 Al(mal)3 groups, and neuronal cell viability was detected by CCK-8 method. Al(mal)3 at 20 μmol·L−1 was selected for subsequent experiments to establish a neuronal network injury model for intervention. The lentivirus infection method was used to transfect miR-29a into neurons, which were divided into mNG, mNG+20 μmol·L−1 Al(mal)3, miR-29a, and miR-29a+20 μmol·L−1 Al(mal)3 groups, and micro-electrode array (MEA) was used to analyze the firing of neuronal network. The expressions of miR-29a and PTEN mRNA in each group were detected by real-time PCR (RT-PCR), and the expression of PTEN protein in each group was detected by Western blotting.
Results The purity of primary mouse hippocampal neurons was greater than 90%, and the viability of the neurons was above 80% in all groups. At 48 h of the designed Al(mal)3 treatments, the changes in spike frequency, burst frequency, network burst frequency, and synchrony index of neurons cultivated on MEA plates in the control group were 207.56%±38.70%, 73.19%±46.43%, 75.42%±33.04%, and 117.13%±15.54%, respectively; the Al(mal)3 groups’ neuronal network electrical activity showed a decreasing trend. Compared with the control group, the spike frequency, burst frequency, network burst frequency, and synchrony index of the 20 and 40 μmol·L−1 Al(mal)3 groups significantly decreased (The changes were 171.70%±28.08%, 49.20%±23.23%, 50.20%±18.18%, and 85.45%±20.30%; 150.68%±26.15%, 43.43%±15.54%, 52.05%±26.31%, and 26.80%±8.29%, respectively, P < 0.05). Compared with the control group (1.00), the miR-29a relative expression levels were significantly decreased in the 20 μmol·L −1 Al(mal)3 group (0.74±0.09) and the 40 μmol·L−1 Al(mal)3 group (0.62±0.12) (P < 0.05); the relative expression levels of PTEN mRNA were significantly increased in the 20 μmol·L−1 Al(mal)3 group (1.32±0.12) and the 40 μmol·L−1 Al(mal)3 group (1.48±0.11) (P < 0.05); the PTEN protein relative expression levels (1.29±0.12 and 1.82±0.10, respectively) were also significantly increased ( P < 0.05). By overexpressing miR-29a in mouse primary hippocampal neurons, the spike frequency, burst frequency, and network burst frequency were significantly higher in the miR-29a group compared with the mNG group (The changes were 252.80%±62.03%, 171.65%±56.30%, and 197.75%±27.12%, respectively, P<0.05). The mNG+20 μmol·L−1 Al(mal)3 group showed a significant decrease in all indicators of neuronal network electrical activity (The changes were 123.28%±47.31%, 66.62%±31.53%, 70.60%±12.48%, and 52.86%±20.26%, respectively, P < 0.05). Compared with the mNG+20 μmol·L −1 Al(mal)3 group, the electrical activity indicators of neuronal network were significantly higher in the miR-29a+20 μmol·L−1 Al(mal)3 group (The changes were 161.41%±42.13%, 101.16%±30.63%, 127.02%±29.58%, and 109.73%±15.61%, respectively, P < 0.05). Compared with the mNG group (1.00), the neuronal PTEN mRNA relative expression (0.67±0.11) and the PTEN protein expression (0.75±0.08) were decreased in the miR-29a group (P < 0.05); the PTEN mRNA relative expression (1.32±0.12) and the PTEN protein relative expression (1.46±0.15) in the mNG+20 μmol·L−1 Al(mal)3 group were increased (P < 0.05). Compared with the mNG+20 μmol·L −1 Al(mal)3 group, the PTEN mRNA relative expression (0.93±0.06) and the PTEN protein relative expression (0.92±0.09) were decreased in the miR-29a+20 μmol·L−1 Al(mal)3 group (P < 0.05).
Conclusion Aluminum significantly inhibits the electrical activity of hippocampal neuronal networks, and miRNA-29a may be involved in the aluminum-induced impairment of hippocampal neuronal network electrical activity by regulating PTEN expression.
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