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首页> 外文期刊>Neural regeneration research >Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe
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Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe

机译：人皮层星形胶质细胞中γ-突触核蛋白的Chimera RNA干扰敲击导致有丝分裂灾难

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Elevated levels of γ-synuclein (γ-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. γ-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized γ-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous γ-syn in primary astrocytes, and the relationship to these two opposing disease states are unknown. γ-Syn is expressed by astrocytes in the human cortex, and to gain a better understanding of the role of endogenous γ-syn, primary human cortical astrocytes were treated with chimera RNA interference (RNAi) targeting γ-syn after release from cell synchronization. Quantitative polymerase chain reaction analysis demonstrated an increase in endogenous γ-syn expression 48 hours after release from cell synchronization, while RNAi reduced γ-syn expression to control levels. Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi γ-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization. To further investigate the consequence of γ-syn knockdown on the astrocytic cell cycle, phosphorylated histone H3 pSer10 (pHH3) and phosphorylated cyclin dependent kinase-2 pTyr15 (pCDK2) levels were observed via western blot analysis. The results revealed an elevated expression of pHH3, but not pCDK2, indicating γ-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours. Subsequently, flow cytometry with propidium iodide determined that increases in apoptosis coincided with γ-syn knockdown. Therefore, γ-syn exerts its effect to allow normal astrocytic progression through the cell cycle, as evidenced by decreased proliferation marker expression, increased pHH3, and mitotic catastrophe after knockdown. In this study, we demonstrated that the knockdown of γ-syn within primary human cortical astrocytes using chimera RNAi leads to cell cycle disruption and apoptosis, indicating an essential role for γ-syn in regulating normal cell division in astrocytes. Therefore, disruption to γ-syn function would influence astrocytic proliferation, and could be an important contributor to neurological diseases.
机译：已经在胶质细胞母细胞瘤的进展中注意到γ-突触核蛋白（γ-SYN）表达的升高，以及诊断出神经变性疾病的患者的脑脊液中。 γ-Syn可以由细胞外Milieu内化或通过人皮质星形胶质细胞内源性表达。内化γ-SYN导致细胞增殖增加，脑衍生的神经营养因子释放和天文学。然而，原发性星形胶质细胞内源性γ-SYN的功能，以及与这两个对立疾病状态的关系是未知的。 γ-Syn由人皮层中的星形胶质表达，并更好地了解内源性γ-SYN的作用，用嵌入细胞同步释放后用嵌合RNA干扰（RNAi）治疗原发性人皮质星形胶质细胞。定量聚合酶链反应分析表明，在从细胞同步释放后48小时内源性γ-SYN表达的增加，而RNAI将γ-SYN表达降低到控制水平。 KI67和5-溴二氧基脲的免疫细胞化学显示嵌合体RNAiγ-SYN敲低24和48小时在细胞同步后24和48小时降低细胞增殖。为了进一步研究星形胶质细胞周期上的γ-SYN敲低的后果，通过蛋白质印迹分析观察到磷酸化的组蛋白H3P3SER10（PHH3）和磷酸化的细胞周期蛋白依赖性激酶-2 pTYR15（PCDK2）水平。结果表明，PHH3的表达升高，但不是PCDK2，表明γ-SYN敲低导致48小时后细胞周期的破坏和染色体压实。随后，具有碘化丙啶的流式细胞术确定凋亡的增加与γ-SYN敲低一致。因此，γ-SYN施加其效果，以允许通过细胞周期允许正常的星形胶质细胞进展，如敲低后的增殖标记表达，pHH3和有丝分裂灾难所证明的。在这项研究中，我们证明，使用Chimera RNai的原发性人体皮质星形胶质细胞内的γ-SYN敲低导致细胞周期破坏和凋亡，表明γ-SIN在半胶质细胞中调节正常细胞分裂中的基本作用。因此，对γ-SYN功能的破坏会影响星形胶质细胞增殖，并且可能成为神经疾病的重要贡献者。

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