PPM1D Silencing by Lentiviral-mediated RNA Interference Inhibits Proliferation and Invasion of Human Glioma Cells
PPM1D Silencing by Lentiviral-mediated RNA Interference Inhibits Proliferation and Invasion of Human Glioma Cells
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DOI：
10.1007/s11596-011-0157-1
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发表时间：
2011-02-01
影响因子：
--
通讯作者：
Yang, Lin
中科院分区：
文献类型：
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作者：
Wang, Peng;Rao, Jing;Yang, Lin
为构建靶向人蛋白磷酸酶1D镁依赖性（PPM 1D）基因的慢病毒shRNA载体，并检测其在人脑胶质瘤中的基因沉默效果，设计并合成了具有特异性短发夹结构的siRNA靶点。将DNA oligo克隆到pFU-GW-iRNA慢病毒表达载体中，然后进行PCR和测序分析以验证构建体。将验证过的质粒转染293 T细胞，制备慢病毒并测定病毒滴度。采用实时定量PCR和Western blot方法检测PPM 1D在胶质瘤细胞中的表达水平。PCR和Western blot分析显示了最佳的干扰靶点，并成功包装了滴度为6 × 108 TU/mL的病毒。病毒感染可在mRNA和蛋白水平上下调人脑胶质瘤细胞中PPM 1D的表达。与对照组相比，PPM 1DmRNA和蛋白表达分别降低76.3%和87.0%。采用流式细胞仪和CCK-8试剂盒检测PPM 1D RNA干扰后人脑胶质瘤细胞的多功能。在U87-MG细胞中，有效下调PPM 1D导致细胞凋亡显著增加，细胞增殖和侵袭能力降低。我们成功地构建了能够在mRNA和蛋白水平上稳定沉默胶质瘤细胞中PPM 1D基因的慢病毒shRNA表达载体。我们的数据提供了证据，证明在神经胶质瘤细胞中PPM 1D沉默后观察到的细胞生长减少至少部分是由于凋亡细胞死亡增加。
To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent (PPM1D) gene and detect its effectiveness of gene silencing in human gliomas, specific siRNA targets with short hairpin frame were designed and synthesized. DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. After the verified plasmids were transfected into 293T cells, the lentivirus was produced and the titer of virus was determined. Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells. PCR and Western blot analyses revealed the optimal interfering target, and the virus with a titer of 6x108 TU/mL was successfully packaged. The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection. The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group. The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8 (CCK-8). Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells. We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells. And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.