SPARC过表达通过调控CPBP/MLKL增强SKM-1细胞对Ara-C的敏感性  

SPARC Overexpression Enhances the Sensitivity of SKM-1 Cells to Ara-C by Regulating CPBP/MLKL

作　　者：梁思敏[1] 周晓佳 蔡铎 周巧[1] 王利[1] LIANG Si-Min;ZHOU Xiao-Jia;CAI Duo;ZHOU Qiao;WANG Li(Department of Hematology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院血液科,重庆400016

出　　处：《中国实验血液学杂志》2022年第5期1508-1514,共7页Journal of Experimental Hematology

摘　　要：目的:探讨SPARC基因过表达对AML-MDS细胞株SKM-1阿糖胞苷(Ara-C)化疗敏感性的影响及调控机制。方法:该实验分为正常SKM-1细胞(对照)、空载(LV-NC)、SPARC过表达(LV-SPARC)、SKM-1细胞+30 ng/ml Ara-C(30ng/ml Ara-C)、LV-NC+30 ng/ml Ara-C和LV-SPARC+30 ng/ml Ara-C共6组。采用CCK-8法检测细胞活性,流式细胞术检测细胞周期分布和凋亡,RT-qPCR检测各组细胞SPARC、CPBP和MLKL的mRNA表达水平,Western blot检测相关蛋白表达水平。结果:SPARC过表达和Ara-C共处理后,细胞活性明显降低,细胞凋亡率明显增高,BCL-2蛋白表达水平显著降低,Bax蛋白表达水平显著增高(P<0.05)。与对照组相比,LV-SPARC+30 ng/ml Ara-C组的细胞周期明显阻滞于S期,CDK2表达水平显著降低,p27^(KIP1)表达水平明显增高(P<0.05)。与LV-SPARC组和30 ng/ml Ara-C组相比,LV-SPARC+30 ng/ml Ara-C组的CPBP和MLKL(p-MLKL)的m RNA和蛋白表达水平明显增高(P<0.05)。SPARC过表达和Ara-C共处理后,p-AKT蛋白表达水平降低,p53蛋白表达水平增高(P<0.05)。结论:SPARC过表达增强了SKM-1细胞对Ara-C的敏感性,促进了细胞周期阻滞和细胞凋亡能力,其机制可能与调控CPBP/MLKL途径相关。Objective: To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. Methods: Subjects were divided into 6groups: SKM-1 cells(Control), Negative control(LV-NC), SPARC overexpression(LV-SPARC), SKM-1 cells+30 ng/ml Ara-C(30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC,CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot.Results: After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2(P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious downregulation of CDK2 and up-regulation of p27^(KIP1)(P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group,the mRNA and protein expression levels of CPBP and MLKL(p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group(P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased(P<0.05). Conclusion: SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.

关 键 词：SPARC基因 阿糖胞苷 SKM-1细胞 CPBP 敏感性 

分 类 号：R733[医药卫生—肿瘤]