Function of UCHL3 in maintaining the survival of FLT3-ITD positive acute myeloid leukemia cells

doi:10.3969/j.issn.1674-8115.2022.10.003

Abstract

Abstract:

Objective ·To explore differently expressed deubiquitinases in acute myeloid leukemia (AML) cells with internal tandem duplications of the FLT3 (FLT3-ITD) gene mutation. Methods ·The expressions of some deubiquitinases were detected by Western blotting after transforming exogenous FLT3-ITD or the use of FLT3-ITD inhibitor. The Cancer Genome Atlas (TCGA) public database was used to analyze the expression of UCHL3 in FLT3-ITD-positive patients and its correlation with the prognosis of AML patients. Certain deubiquitinase was knocked down by short hairpin RNA (shRNA) in AML cell lines. Cell counting, CCK-8 assay, flow cytometry and wright stain were used to detect the effect of certain deubiquitinase on the function of AML cells and the combined effect of knockdown of corresponding deubiquitinase or using its inhibitor with all-trans-retinoic acid (ATRA). Results ·Expression of FLT3-ITD in Ba/F3 cells significantly induced the protein expression of UCHL3, while inhibition of FLT3-ITD activity in MOLM-13 and MV4-11 cell lines significantly inhibited the expression of UCHL3 in a time- and concentration-dependent manner. TCGA database analysis showed that there was a significant positive correlation between the expression of FLT3 and the expression of UCHL3 in AML (P=0.000), and a significant negative correlation between the expression of UCHL3 and the survival rate of AML patients (P=0.016). UCHL3 knockdown inhibited proliferation and promoted apoptosis of MOLM-13 and MV4-11 cells, and the expression of differentiation-related transcription factors PU.1 and C/EBPβ were significantly increased. Knockdown of UCHL3 significantly enhanced the apoptosis of MOLM-13 and MV4-11 cells induced by 100 nmol/L ATRA, and the apoptosis-related caspase proteins PARP1, caspase 9 and caspase 3 were significantly cleaved. Meanwhile, UCHL3 knockdown cells showed more features of small nuclear volume, nuclear depression, and rod-shaped or irregular cells after ATRA treatment. The dose-response curves of ATRA and TCID detected by CCK-8 assay showed that UCHL3 inhibitor TCID and ATRA synergistic inhibited FLT3-ITD-positive AML cells survival. Conclusion ·FLT3-ITD up-regulates UCHL3 expression and promotes the survival of AML cells. UCHL3 is negatively correlated with the prognosis of AML patients. Knockdown or inhibition of UCHL3 combines with ATRA to inhibit the survival of FLT3-ITD-positive AML cells.

Key words: ubiquitin carboxyl-terminal hydrolase L3 (UCHL3), acute myeloid leukemia (AML), FMS-like tyrosine kinase 3 (FLT3), FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), all-trans retinoic acid (ATRA)

CLC Number:

R733.71

HU Jiacheng, ZHU Qian, WANG Jiaqi, WU Yingli, LEI Hu. Function of UCHL3 in maintaining the survival of FLT3-ITD positive acute myeloid leukemia cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(10): 1383-1393.

Figures/Tables 7

Fig 1 FLT3-ITD up-regulates UCHL3 expression

Fig 2 Correlation between expression of UCHL3 and AML prognosis

Fig 3 Effect of UCHL3 knockdown on the proliferation of FLT3-ITD-positive AML cell lines

Fig 4 Effects of UCHL3 knockdown on apoptosis and differentiation of FLT3-ITD-positive AML cell lines

Fig 5 Effect of UCHL3 knockdown combining with ATRA on apoptosis of FLT3-ITD-positive AML cell lines

Fig 6 Effects of ATRA after UCHL3 knockdown on differentiation of FLT3-ITD-positive AML cells

Fig 7 Synergistic effects of TCID and ATRA on the viability of MV4-11 and MOLM-13 cells

TrendMD

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