摘要
[目的]探讨胶原三螺旋重复蛋白1(CTHRC1)对白血病K562细胞增殖、凋亡和周期的影响。[方法]通过CAMOIP和TARGET数据库分析CTHRC1在AML中的表达情况;通过单细胞数据集GSE116256验证CTHRC1的异质性表达;通过慢病毒包装与感染构建CTHRC1干扰和过表达稳转K562细胞系;通过流式细胞术和细胞计数实验检测CTHRC1表达对K562细胞增殖、凋亡和周期的影响。[结果]CAMOIP数据库与TARGET数据库均表明CTHRC1在AML中表达上调(P<0.001);TARGET数据库生存分析表明CTHRC1高表达患者生存较低表达组差(P=0.006);Kmplot数据库生存分析结果也表明CTHRC1高表达患者生存较低表达组差(P=0.018);单细胞分析表明CTHRC1主要在白血病相关B细胞中表达;蛋白检测结果显示CTHRC1干扰组与质粒空载组相比表达明显下调(P=0.005,t=10.39),过表达组与过表达组空载组相比明显上调(P<0.001,t=15.91);mRNA水平检测结果显示干扰组与干扰质粒空载组相比明显下调(P<0.001,t=19.05),过表达组与过表达组空载组相比明显上调(P=0.0014,t=7.893);细胞增殖结果表明CTHRC1下调抑制K562细胞增殖(P<0.001),过表达促进K562细胞增殖(P=0.042);流式细胞术检测细胞凋亡结果表明:与正常组相比,敲低CTHRC1促进K562细胞凋亡(P<0.001,t=36.79),过表达CTHRC1抑制K562细胞凋亡(P<0.001,t=64.93);流式细胞术检测细胞周期结果表明:CTHRC1下调后K562细胞周期S期(P<0.001,t=14.78)和G_(2)/M(P=0.008,t=9.074)期被抑制,G_(0)/G_(1)期升高(P<0.001,t=23.57),CTHRC1过表达后细胞周期S期(P<0.001,t=14.26)和G_(2)/M期(P=0.0207,t=3.705)被抑制,G_(0)/G_(1)期降低(P=0.001,t=23.57),细胞周期S期比例升高。[结论]CTHRC1为白血病不良预后指标,主要在白血病B细胞中表达,可介导K562细胞增殖、凋亡和周期,这些发现为白血病的治疗提供了理论基础。
[Objective]To investigate the effects of collagen triple helix repeat protein 1(CTHRCI)on the proliferation,apop-tosis and cycle of leukaemia K562 cells.[Method]CTHRC1 expression in AML was analysed by CAMOIP and TARGET data-bases;heterogeneous expression of CTHRC1 was verified by the single-cell dataset GSE116256;CTHRC1-disrupting and overexpressing stably-transfected K562 cell lines were constructed by lentiviral packaging and infection;and the effect of CTHRC1 expression on the proliferation,apoptosis and cycle of K562 cells was examined by flow cytometry and cell counting experiments.The effects of CTHRC1 expression on the proliferation,apoptosis and cell cycle of K562 cells were detected by flow cytometry and cell counting assay.[Result]Both CAMOIP database and TARCET database showed that CTHRCI expression was up-regulated in AML(P<0.001);survival analysis of TARGET database showed that patients with high CTHRC1 ex-pression had a worse survial than those with low expression(P=0.006);Kmplot database survival analysis also indicated the same(P=0.018);single-cell analysis showed that CTHRC1 was mainly expressed in leukaemia-associated B cells;pro-tein assay showed that the expression of CTHRC1 was significantly down-regulated in the interfering group compared with that of the plasmid-empty group(P=0.005,t=10.39),and significantly up-regulated in the overexpression group compared with that of the overexpression group in the plasmid-empty group(P<0.001,t=15.91);and the mRNA level assay results showed that the expression in the interference group was significantly down-regulated compared with that in the interference plasmid empty group.The results of mRNA levels showed that the interference group was significantly down-regulated com-pared with the interference plasmid empty group(P<0.001,t=19.05),and the overexpression group was significantly up-regulated compared with the overexpression group empty group(P=0.0014,t=7.893).The results of cell proliferation showed that CTHRC1 down-regulation inhibited K562 cell proliferation(P<0.001),and overexpression promoted K562 cell proliferation(P=0.042);the results of apoptosis detected by flow cytometry showed that knockdown of CTHRC1 promoted K562 cell apoptosis compared with the normal group(P<0.001,t=36.79),and overexpression of CTHRC1 inhibited K562 cell apoptosis(P<0.001,t=64.93),and the cell cycle results of flow cytometry showed that the S-phase(P<0.001,t=14.78)and G_(2)/M-phase(P=0.008,t=9.074)were inhibited after CTHRC1 was down-regulated,and the G_(0)/G_(1)-phase was increased(P<0.001,t=23.57).The cell cycle S phase(P<0.001,t=14.26)and G_(2)/M phase(P=0.0207,t=3.705)were suppressed,and G_(0)/G_(1) phase was decreased(P=0.001,t=23.57)after CTHRC1 overexpression,and the cell cycle S phase ratio was elevated.[Conclusion] CTHRCI is a poor prognostic indicator for leukaemia and is mainly expressed in leukaemic B cells,which can regulate the proliferation,apoptosis and cycle of K562 cells.These findings provide a theoretical basis for the treatment of leukaemia.
作者
冯小云
秦玉凤
张鹏
FENG Xiaoyun;QIN Yufeng;ZHANG Peng(Center for Tissue Engineering and Stem Cells,Guizhou Medical University,Department of Biology,Guizhou Medical University,Guiyang 550004;Department of Prosthetics,Affiliated Stomatological Hospital of Guizhou Medical University,Guiyang 550004,China)
出处
《生物技术》
2025年第4期465-475,共11页
Biotechnology
基金
国家自然科学基金地区科学基金项目(81960036)
贵州省科技计划项目(黔科合基础-ZK[2021]重点005)
贵州省教育厅高等学校血液疾病地中海贫血及白血病治疗生物药的开发及机制研究创新团队(黔教技[2023]068号)。
