NCTD通过调控PPP5C影响人白血病细胞增殖和凋亡的机制研究

Effects and mechanism of NCTD on proliferation and apoptosis of human leukemic cells by targeting PPP5C

目的研究去甲斑蝥素(norcantharidin,NCTD)通过调控磷酸蛋白磷酸酶催化亚基5C(protein phosphatase 5 catalytic subunit,PPP5C)对人白血病NB4、K562细胞增殖、凋亡能力的影响及机制的初步研究。方法体外培养NB4、K562细胞,电转PC3.1和PPP5C-PC3.1质粒至NB4、K562细胞,遗传霉素(geneticin,G418)筛选NB4、K562稳转细胞系。Western blot和RT-qPCR实验检测PPP5C蛋白和mRNA表达水平。采用CCK-8、迁移实验、Live&Dead^(TM)动物细胞活力/毒性检测试剂盒分别检测NB4、K562细胞的增殖能力、迁移能力和死细胞、活细胞的数量。将NB4、K562稳转细胞分为0μg/mL NCTD组和不同浓度NCTD组,分别用含有0、8、16、32μg/mL NCTD的1640培养基培养;Live&Dead^(TM)动物细胞活力/毒性检测试剂盒检测死细胞率并对细胞形态进行拍照;Western blot检测各组细胞caspase 3、Cleaved caspase 3、JNK、p-JNK、p38、p-p38和α-Tubulin蛋白表达水平。结果NB4、K562细胞转染后PPP5C表达水平显著提高,细胞增殖能力、迁移能力、抗凋亡能力显著增强;与0μg/mL NCTD组相比,NCTD各浓度组会促进细胞凋亡,且呈剂量依赖性;PPP5C过表达拮抗NCTD对白血病细胞的杀伤作用;机制研究发现PPP5C通过去磷酸化修饰降低p-JNK的蛋白水平进而调控与细胞凋亡相关蛋白Cleaved caspase 3的表达。结论NCTD能够通过调控PPP5C分子促进NB4、K562细胞凋亡,抑制细胞的增殖。

Objective To study the effects and mechanism of norcantharidin(NCTD)on proliferation and apoptosis of NB4 and K562 human leukemic cells by regulating phosphoprotein phosphatase 5 catalytic(PPP5C).Methods PC3.1 and PPP5C-PC3.1 plasmids were electroporated into NB4 and K562 cells.Stable NB4 and K562 cell lines were selected with geneticin(G418).Protein and mRNA expression levels of PPP5C were measured by Western blot and RT-qPCR,respectively.Proliferation,migration,and apoptosis of NB4 and K562 cells were determined by a CCK-8 assay,transwell assay,and Live&Dead^(TM) animal cell viability/toxicity detection kit,respectively.NB4 and K562 cells were divided into 0μg/mL NCTD group and various NCTD dose groups,and cultured in RPMI 1640 medium containing 0,8,16,or 32μg/ml NCTD.The Live&Dead^(TM) animal cell viability/toxicity detection kit measured the numbers of dead and live cells,and cell morphology was observed under a microscope.Western blot was used to measure protein expression levels of caspase 3,Cleaved caspase 3,JNK,p-JNK,p38,p-p38,andα-Tubulin.Results Proliferation,migration,and apoptosis of NB4 and K562 cells were enhanced by overexpression of PPP5C.Compared with 0μg/mL NCTD group,NCTD promoted apoptosis in a dose-dependent manner.PPP5C overexpression antagonized the killing effect of NCTD on leukemic cells.Mechanistic investigations showed that PPP5C reduced the protein level of p-JNK by dephosphorylating and regulating the expression of apoptosis-related protein Cleaved caspase 3.Conclusions NCTD promotes apoptosis of NB4 and K562 cells and inhibits their proliferation by inhibiting PPP5C.