Objective: To investigate the effect of PPP2R5C to the activity of Molt-4 cells in childhood acute T lymphocytic leukemia and its mechanism.
Methods: The small interfering RNA (siRNA) technology targeting PPP2R5C gene was used to down-regulate the expression of PPP2R5C in Molt-4 cells. At the same time, a blank control group, a negative control group and a 17-DMAG group were set up. The cells in the negative control group were transfected with siRNA-NC, the cells in 17-DMAG group were treated with the HSP90 inhibitor 17-DMAG at a final concentration of 6.4 μmol/L for 48 h. Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot were used to detect transfection efficiency; CCK-8 method was used to detect the proliferation activity of the cells in each group, EdU was used to detect the proliferation level of the cells in each group, flow cytometry was used to detect the cell cycle distribution ratio of the cells in each group, Annexin V-FITC/PI staining was used to detect the apoptosis of the cell, RT-qPCR and Western blot were used to detect the expression changes of heat shock protein 90 (HSP90) and glucocorticoid receptor (GR) of the cells in each group.
Results: After Molt-4 cells were transfected with siRNA-PPP2R5C, the expression of PPP2R5C mRNA and protein in the cells were down-regulated significantly compared with those in the blank control group and the si-NC group (P<0.05); compared with cells in the blank control group and the si-NC group, the proliferation activity of the cells in the siRNA-PPP2R5C group and the 17-DMAG group significantly decreased (P<0.05), and the rate of EdU positive cells was significantly reduced (P<0.05); the proportion of the cells in G1 phase decreased while the proportion of the cells in G2 phase increased (P<0.05), the apoptosis rate of the cells also increased significantly (P<0.05); in addition, the expression of PPP2R5C mRNA and protein of the cells in siRNA-PPP2R5C group was significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05). The expressions of PPP2R5C mRNA and protein in the 17-DMAG group were also significantly down-regulated compared with those in the blank control group and si-NC group (P<0.05).
Conclusion: Down-regulation of PPP2R5C gene expression can inhibit Molt-4 cell activity in childhood acute T lymphocytic leukemia, block the cells in G2 phase, and promote cell apoptosis, the mechanism may be related to the inhibition of HSP90-GR signaling pathway.
题目: PPP2R5C与儿童急性T淋巴细胞白血病Molt-4细胞活性 及Hsp90-GR信号关系的研究.
目的: 探讨PPP2R5C对儿童急性T淋巴细胞白血病Molt-4细胞活性的影响及其相关机制.
方法: 通过靶向PPP2R5C基因的小干扰RNA（siRNA）技术下调Molt-4细胞中PPP2R5C的表达，同时设置空白对照组、阴性对照组及17-DMAG组，其中阴性对照组转染siRNA-NC，17-DMAG组使用终浓度为6.4 μmol/L的热休克蛋白90（HSP90）抑制剂17-DMAG处理细胞48 h，采用实时荧光定量PCR（RT-qPCR）和免疫印迹法（Western blot）检测转染效率；CCK-8法检测各组细胞增殖活性，EdU检测各组细胞增殖水平，流式细胞术检测各组细胞周期分布比例，Annexin V-FITC/PI染色检测细胞凋亡，RT-qPCR和Western blot检测各组细胞中HSP90和糖皮质激素受体（GR）的表达变化.
结果: Molt-4细胞转染siRNA-PPP2R5C后，细胞中PPP2R5C mRNA与蛋白表达均明显下调，与空白对照组和si-NC组相比有显著性差异（P<0.05）；相比空白对照组和si-NC组，siRNA-PPP2R5C组和17-DMAG组细胞的增殖活性明显降低（P<0.05），EdU阳性细胞率明显减少（P<0.05），G1期细胞比例减少而G2期细胞比例增加（P<0.05），细胞凋亡率也明显增加（P<0.05）；此外，siRNA-PPP2R5C组细胞中，PPP2R5C mRNA与蛋白表达较空白对照组和si-NC组明显下调（P<0.05），17-DMAG组细胞中PPP2R5C mRNA与蛋白表达较空白对照组和si-NC组也明显下调（P<0.05）.
结论: 下调PPP2R5C基因表达可抑制儿童急性T淋巴细胞白血病Molt-4细胞的活性，使细胞发生G2期阻滞，并促进细胞凋亡，其机制可能与抑制Hsp90-GR信号途径相关.
Keywords: PPP2R5C; cell viability; childhood acute T lymphocytic leukemia; glucocorticoid receptor; heat shock protein 90.