摘要
目的探讨C型凝集素结构域家族5成员A(CLEC5A)基因对白血病细胞系THP-1和K562细胞增殖、凋亡和周期进程的影响及其可能的作用机制。方法通过基因表达谱交互分析(GEPIA)数据库分析白血病患者中CLEC5A的表达水平。采用包装了CLEC5A过表达质粒的慢病毒感染THP-1和K562细胞以过表达CLEC5A,采用小干扰RNA(siRNA)瞬时转染THP-1和K562细胞以敲低CLEC5A。采用CCK-8实验与EdU实验检测细胞的增殖能力,流式细胞术检测细胞周期及过氧化氢(H_(2)O_(2))刺激条件下的细胞凋亡。通过转录组测序和通路富集分析过表达CLEC5A基因后,上调或下调表达的基因显著富集的信号通路。采用Western blot实验检测AKT/mTOR和p53信号通路中相关蛋白的表达水平。结果CLEC5A在人白血病骨髓组织中的表达水平显著高于健康人群。敲低CLEC5A可显著抑制THP-1和K562细胞的增殖(P<0.01)和S期进程(P<0.05),并促进H_(2)O_(2)刺激条件下的细胞凋亡(P<0.001);过表达CLEC5A则显著促进THP-1和K562细胞的增殖(P<0.001)和S期进程(P<0.01),并抑制H_(2)O_(2)刺激条件下的细胞凋亡(P<0.01)。过表达CLEC5A后上调表达的基因显著富集于AKT1/mTOR等信号通路;而下调表达的基因显著富集于细胞周期等信号通路。且CLEC5A可显著抑制BAX和p53基因的表达,显著促进BCL-2基因的表达和AKT1、mTOR蛋白的磷酸化水平。结论CLEC5A可促进白血病细胞系THP-1和K562的细胞增殖和周期进程,抑制细胞凋亡,其作用机制可能与AKT/mTOR和p53信号通路相关。
Objective To investigate the effects of C-type lectin domain family 5,member A(CLEC5A)on the proliferation,apoptosis,and cell cycle of leukemia cell lines THP-1 and K562,and the underlying mechanism.Methods The expression of CLEC5A in leukemia patients was investigated in the GEPIA database.Recombined plasmid containing CLEC5A was transfected into THP-1 and K562 cells for overexpression of CLEC5A.Small interfering RNA(siRNA)was used to knock down the endogenous CLEC5A in leukemia cells.CCK-8 and EdU assays were used to assess the leukemia cells proliferation.Flow cytometry was used to assess cell cycle.Flow cytometry was used to assess cell apoptosis under hydrogen peroxide(H_(2)O_(2))stress.The RNA sequencing(RNA-seq)and pathway enrichment analysis were used to analyze the signal pathways of significant enrichment of up-regulated or down-regulated genes after knocking down CLEC5A gene.Protein expression levels of several members in AKT1/mTOR and p53 signaling pathways were detected by Western blot assays.Results CLEC5A was significantly up-regulated in bone marrow tissues of leukemia patients compared to the matched non-tumor tissues of healthy individuals.Knockdown of CLEC5A significantly reduced the proliferation(all P<0.01)and S phase progression(all P<0.05),and increased the apoptosis(all P<0.001)under H_(2)O_(2)stress,in THP-1 and K562 cells.Conversely,overexpression of CLEC5A significantly increased the proliferation(all P<0.001)and S phase progression(all P<0.01),and reduced the apoptosis(all P<0.01)under H_(2)O_(2)stress,in THP-1 and K562 cells.The up-regulated genes were significantly enriched in AKT1-mTOR and other signal pathways after knocking down CLEC5A,while the down-regulated genes were significantly enriched in cell cycle signal pathways.CLEC5A in leukemia cells significantly reduced the genes expression levels of BAX and p53,and significantly induced the gene expression levels of BCL-2 and phosphorylation levels of AKT1 and mTOR proteins.Conclusion CLEC5A increases the cell cycle and proliferation and inhibits cells apoptosis in THP-1 and K562 cells,and the mechanism may be related to activating the AKT/mTOR and p53 signaling pathways.
作者
丁书琴
查丹彤
齐欣
杨爱清
周钢桥
Ding Shuqin;Zha Dantong;Qi Xin;Yang Aiqing;Zhou Gangqiao(School of Life Sciences,Anhui Medical University,Hefei 230032;State Key Laboratory of Proteomics,National Center for Protein Sciences,Beijing Institute of Radiation Medicine,Beijing 100850;Medical College,Guizhou University,Guiyang 550025)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第10期1613-1621,共9页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:8190061543)。
