Effects of TRAF6 Over-Expression on Autophagic Activity of Human Acute Myeloid Leukemia Cells
TANG Yuting, TAO Yao, WANG Lu, YANG Liyuan, LEI Li, JING Yipei, JIANG Xueke, JIN Hongjun, ZHANG Ling*
This work was to investigate the effects of TRAF6 (tumor necrosis factor receptor-associated factor 6) overexpression on autophagic activity of human AML (acute myeloid leukemia) cells. The expression levels of TRAF6 mRNA in AML patients derived from GEO (gene expression omnibus) database were analyzed. The relationship between the expression of TRAF6 and the clinical prognosis of AML patients were explored based on TCGA (The Cancer Genome Atlas) database. The TRAF6 recombination plasmids were transfected into two human acute myeloid leukemia cell lines (KG-1a and THP-1). Autophagy activator (Rapamycin) and two different autophagy inhibitors, 3-MA (3-methyladenine) and Baf-A1 (bafilomycin A1) were used to treat the leukemic cells. The effects of TRAF6 overexpression on the mRNA and protein levels of autophagic markers LC3 and p62 in leukemic cells were determined by qRT-PCR and Western blot techniques, respectively. The LC3 puncta was determined by immunofluorescence. The apoptosis rate was detected by flow cytometry. The cell proliferation activity in vitro was evaluated by CCK-8 assay. The results showed that the expression levels of TRAF6 mRNA in AML patients were higher than those observed in normal controls (P<0.01). Additionally, high expression of TRAF6 showed a trend towards lower OS (overall survival) and EFS (event-free survival) (P=0.01) in AML patients, than that in low expression of TRAF6 group. The expression levels of TRAF6 mRNA and protein were significantly increased in TRAF6 overexpression group compared with those in the control group (P<0.05). Rapamycin treatment enhanced the autophagic activity of leukemic cells. Importantly, TRAF6 overexpression significantly upregulated LC3 mRNA and LC3II protein levels (P<0.05), downregulated p62 mRNA and protein levels and increased the accumulation of LC3 puncta in leukemic cells (P<0.05). Notably, Baf-A1 treatment significantly increased LC3II protein levels in TRAF6-enforced cells (P<0.05). Exposure to 3-MA significantly downregulated LC3II protein levels and upregulated p62 protein levels in TRAF6-enforced expression cells (P<0.05). Finally, the apoptosis rate was significantly decreased and the cell proliferation was significantly enhanced in TRAF6-enforced group (P<0.001), while the treatment of 3-MA attenuated the ability of TRAF6 overexpression mediated growth advantage (P<0.001). Abovementioned results suggest that TRAF6 high expression enhances the autophagic activity and promotes leukemic cell growth.
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