PPP2R5C蛋白作为诱导CML细胞凋亡分子靶标的机制研究

The abnormal expression of PPP2R5C gene was closely related the occurrence of chronic myeloid leukemia (CML). Our previous study found that PPP2R5C-siRNA （small interfering RNA）can induce the apoptosis of CML cell lines (imatinib-resistant or imatinib-sensitive chronic myeloid leukemia cells). Global gene expression profiling analysis indicated that PPP2R5C might exert these effects via targeting TRAIL signal pathway. Herein, we will further explore PPP2R5C gene by using gene transfer, RNA interference and regulate the expression of TRAIL in imatinib-resistant CML cell line and imatinib-resistant primary CML cells. We will identify the correlation between changeable PPP2R5C and TRAIL apoptosis. We will detect the changes of the key molecules in TRAIL signal pathway such as TRAIL, TRAIL's receptor and BCL2L1 by performing quantitative RT-PCR and Western Blotting. Also we will compare the sensitive of TRAIL apoptosis between imatinib-resistant and imatinib-sensitive CML cells. Upon completing this project, we expect that our findings can be confirmed the effect and molecular mechanism in CML cells by downregulating PPP2R5C. In addition, PPP2R5C and TRAIL may provide a new therapeutic target or combination treatment way in imatinib-sensitive CML.

PPP2R5C基因异常表达与慢性粒细胞白血病（CML）发生密切相关。我们前期研究发现PPP2R5C-siRNA(小干扰RNA)可以诱导CML细胞株（伊马替尼耐药细胞株K562R及K562）凋亡，基因芯片结果分析推测其可能通过TRAIL相关的信号通路发挥作用。本研究拟进一步利用基因转染和RNA干扰技术改变CML细胞株及原代CML细胞（初发未治CML及伊马替尼耐药CML患者）中PPP2R5C基因，并调节TRAIL的表达，明确:1）PPP2R5C的改变与TRAIL凋亡通路的相关性，2）分析TRAIL通路关键分子TRAIL及其受体、BCL2L1等的改变；3）比较耐药及非耐药CML细胞对TRAIL诱导的凋亡敏感性。本项目的实施将明确沉默CML细胞PPP2R5C蛋白与CML细胞凋亡的作用及分子机制，为CML尤其是耐药CML的治疗提供新靶点或联合用药新方法。

通过构建 PPP2R5C基因过表达慢病毒体系及PPP2R5C-shRNA慢病毒体系，改变慢性粒细胞性白血病伊马替尼敏感和耐药细胞株（K562, K562R）中PPP2R5C 基因的表达，并调节TRAIL的表达，明确:1）PPP2R5C的改变与TRAIL凋亡通路的相关性，2）分析TRAIL通路相关基因的表达改变；3）比较耐药及非耐药CML细胞对TRAILt通路的不同影响。初步分析下调CML细胞PPP2R5C基因表达细胞凋亡的作用及分子机制，为耐药CML的治疗提供新靶点或联合用药新方法。方法：构建转染PPP2R5C重组质粒及PPP2R5C-shRNA，核转染K562及K562R细胞株；荧光实时定量 PCR 检测各组细胞PPP2RC及TRAIL通路相关基因表达量mRNA的表达水平；流式细胞术检测细胞凋亡；Western blot检测K562各组PPP2R5C蛋白及TRAIL通路相关蛋白的合成情况；结果：1.慢病毒体系的转染，能稳定高效地感染细胞，转染效率均在80%以上,经过mRNA与蛋白水平验证其稳定高效表达。K562、K562R细胞株，PPP2R5C上调和下调，各转染组与对照组相比，凋亡率均增加；TRAIL通路多个相关基因出现了表达量的改变；蛋白合成量的改变基本与表达发生改变的基因相符。结论：PPP2R5C表达模式的改变，直接影响CML细胞凋亡，细胞凋亡的发生，与TRAIL通路多个基因的协调作用有关联。