RNAi抑制PIAS3表达对胶质瘤U251细胞增殖和凋亡的影响

  目的   构建针对PIAS3的RNAi质粒载体，初步探讨抑制PIAS3表达对胶质瘤U251细胞增殖和凋亡的影响。   方法   目的:方法:构建3个RNAi载体，用脂质体法将其转染胶质瘤细胞系CHG-5，通过半定量RT-PCR筛选干扰效率最高的重组RNAi质粒。将该RNAi质粒转染体外培养的人胶质瘤U251细胞株，用半定量RT-PCR和Western blot检测PIAS3的表达。Annexin V2 FITC和PI双染流式细胞术检测细胞凋亡，流式细胞术检测细胞增殖周期变化。   结果   转染干扰质粒的细胞株PIAS3基因和蛋白表达均有减弱。流式细胞仪分析，抑制PIAS3表达能够诱导胶质瘤U251细胞拮抗凋亡（P < 0.01），同时出现细胞周期改变，表现为G2期细胞比例升高，S期细胞比例降低，具有显著性差异（P < 0.05）。   结论   下调PIAS3基因表达使U251阻滞在G2期，拮抗细胞凋亡。  

Effects of PIAS3 silencing by RNAi on the proliferation and apoptosis of U251 glioma cells in vitro

  Objective   To observe the effect of the proteininhibitor of activated STAT 3 (PIAS3) on the proliferation and apoptosis of U251 glioma cells after PIAS3 expression was inhibited by RNAi.   Methods   Three RNAi expression vectorstargeting PIAS3 were constructed and transfected into CHG-5 cells by liposomein vitro. The most efficient RNAi vector was subsequently selected by examiningthe mRNA expressions of PIAS3 in the transfected cells by semi-quantitativeRT-PCR. The selected RNAi vector was then transfected into U251 cells. After 48h of transfection, the mRNA and protein expressions of PIAS3 in glioma cellswere examined by semi-quantitative RT-PCR and western blot. Apoptosis wasobserved by flow cytometry using a double-staining method with FITC-conjugatedannexin V and PI. Flow cytometry was also applied in cell cycle assay.   Results   RNAidownregulated the mRNA (P < 0.01) and protein (P < 0.01) expressionsof PIAS3 in transfected cells.RNAi promoted the resistance of U251 cells to apoptosisand subsequently altered the cell cycle. A high percentage of G2 phaseand a low percentage of Sphase were observed in U251 cells.   Conclusion   The downregulation of PIAS3arrested U251 cells in the G2 phase andinduced the resistance of U251 cells to apoptosis.