雷帕霉素诱导急性T淋巴白血病细胞自噬分子机制的研究  

Study on the molecular mechanism of rapamycin⁃induced autophagy in acute T lymphoblastic leukemia cells

作　　者：许兰 黄笠纹 肖亦舒 刘春亚 杜乐 任立成[1] XU Lan;HUANG Li-wen;XIAO Yi-shu;LIU Chu-nya;DU Le;REN Li-cheng(Department of Biology,Hainan Medical University,Haikou 571199,China)

机构地区：[1]海南医学院生物学教研室,海南海口571199

出　　处：《海南医学院学报》2023年第9期652-659,共8页Journal of Hainan Medical University

基　　金：国家自然科学基金项目(31660318);海南省自然科学基金高层次人才项目(820RC638);校级研究生创新科研课题(HYYS2020-10)。

摘　　要：目的:研究不同浓度雷帕霉素对急性白血病CD4+T-Jurkat细胞增殖的影响,并从自噬方面探讨其作用机制。方法:MTT法检测不同浓度雷帕霉素对细胞增殖的影响;流式细胞术检测细胞凋亡率和周期阻滞;吖啶橙染色观察自噬溶酶体的变化;转录组测序数据分析自噬相关基因的差异表达;RT-qPCR和Western Blot实验检测自噬相关基因的转录和蛋白质表达水平;FAIRE-qPCR分析自噬基因启动子区的染色质可及性状态。结果:与对照组相比,雷帕霉素呈浓度和时间依赖性方式抑制Jurkat细胞的增殖;并将细胞周期阻滞在G0/G1期,但不能有效诱导细胞凋亡。通过吖啶橙染色观察,给药后可见自噬溶酶体的数量增加。RNA-seq数据显示,雷帕霉素可以显著影响自噬相关基因的转录水平。不同浓度雷帕霉素作用于Jurkat细胞48 h后,10 nmol/L和20 nmol/L浓度的雷帕霉素可上调ULK1、ATG13、ATG16L2、PI3K3R1、Raptor基因的表达,下调MAPK1基因的表达;50 nmol/L时,下调各基因的表达水平。自噬基因启动子区域染色质可及性也随之发生改变,与基因表达变化趋势基本一致。结论:雷帕霉素能够抑制Jurkat细胞的增殖,阻滞细胞周期,诱导细胞发生自噬;较低浓度的雷帕霉素促进自噬相关基因的表达,高浓度则抑制其表达。Objective:To study the effect of different concentrations of rapamycin on the proliferation of acute leukemia CD4+T‑Jurkat cells,and to explore its mechanism from the aspect of autophagy.Methods:The effect of different concentrations of rapamycin on cell proliferation was detected by MTT assay;Apoptosis rate and cell cycle arrest were detected by flow cytometry;The changes of autophagic lysosomes were observed by acridine orange staining;Transcriptome sequencing data were used to analyze the differential expression of autophagy‑related genes;The transcription and protein expression levels of autophagy‑related genes were detected by RT‑qPCR and Western Blot;The chromatin accessibility of the promoter region of autophagy gene was analyzed by FAIRE‑qPCR.Results:Compared with the control group,rapamycin inhibited the proliferation of Jurkat cells in a concentration‑and time‑dependent manner.The cell cycle was arrested in G0/G1 phase,but it could not effectively induce apopto‑sis.Observed by acridine orange staining,the number of autophagic lysosomes increased after administration.RNA‑seq data showed that rapamycin could significantly affect the transcription level of autophagy‑related genes.After Jurkat cells were treated with different concentrations of rapamycin for 48 h,10 nmol/L and 20 nmol/L rapamycin could up‑regulate the expression of ULK1,ATG13,ATG16L2,PI3K3R1,Raptor genes and down‑regulate the expression of MAPK1 gene.At 50 nmol/L,the expression levels of each gene were down‑regulated.The chromatin accessibility of the autophagy gene promoter region also changed,which was basically consistent with the trend of gene expression.Conclusion:Rapamycin can inhibit the proliferation of Jurkat cells,block cell cycle and induce autophagy.Low concentrations of rapamycin promoted the expression of autophagy‑related genes,while high concentrations inhibited their expression.

关 键 词：雷帕霉素 自噬基因 细胞周期 基因表达 染色质可及性 

分 类 号：R733.71[医药卫生—肿瘤]