摘要
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 apoptosis.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 nM and 20 nM rapamycin could up-regulate the expression of ULK1,ATG13,ATG16L2,PI3K3R1,Raptor genes and down-regulate the expression of MAPK1 gene.At 50 nM,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.
基金
National Natural Science Foundation of China(No.31660318)
Natural Science Foundation of Hainan Province(No.820RC638)
The Innovative research projects for graduates of Hainan Medical University(No.HYYS2020-10)。
