Wang Peng, Rao Jing, Yang Haifeng, Zhao Hongyang, Yang Lin
Department of Neurosurgery, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510080, China.
Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2011 Feb;31(1):94-99. doi: 10.1007/s11596-011-0157-1. Epub 2011 Feb 19.
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 6×10(8) 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.
构建靶向人镁依赖蛋白磷酸酶1D（PPM1D）基因的慢病毒shRNA载体，并检测其在人胶质瘤中基因沉默的有效性。设计并合成具有短发夹结构的特异性siRNA靶点。将DNA寡核苷酸克隆到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。将经验证的质粒转染到293T细胞中，产生慢病毒并测定病毒滴度。进行实时定量PCR和蛋白质印迹以检测感染的胶质瘤细胞中PPM1D的表达水平。PCR和蛋白质印迹分析揭示了最佳干扰靶点，成功包装出滴度为6×10(8) TU/mL的病毒。通过病毒感染在mRNA和蛋白质水平上敲低了人胶质瘤细胞中PPM1D的表达。与对照组相比，PPM1D mRNA和蛋白质的表达分别降低了76.3%和87.0%。通过流式细胞术和细胞计数试剂盒-8（CCK-8）检测PPM1D RNA干扰后人胶质瘤细胞的多种功能。PPM1D的有效下调导致U87-MG细胞中细胞凋亡显著增加，细胞增殖和侵袭潜能降低。我们成功构建了能够在胶质瘤细胞中在mRNA和蛋白质水平上稳定沉默PPM1D基因的慢病毒shRNA表达载体。我们的数据表明，胶质瘤细胞中PPM1D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所致。
