Upregulation of miR-142-3p Improves Drug Sensitivity of Acute Myelogenous Leukemia through Reducing P-Glycoprotein and Repressing Autophagy by Targeting.

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Upregulation of miR-142-3p Improves Drug Sensitivity of Acute Myelogenous Leukemia through Reducing P-Glycoprotein and Repressing Autophagy by Targeting HMGB1 Yuan Zhang, Yufeng Liu, Xueju Xu Translational Oncology Volume 10, Issue 3, Pages 410-418 (June 2017) DOI: 10.1016/j.tranon.2017.03.003 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Expression levels of miR-142-3p and HMGB1 in PBMCs from pediatric AML patients. qRT-PCR assay was performed to examine the expressions of miR-142-3p (A) and HMGB1 mRNA (B) in 23 pediatric AML PBMCs and 15 normal PBMCs. (C) The level of HMGB1 in 23 pediatric AML PBMCs and 15 normal PBMCs was determined by Western blot. (D) A negative correlation between miR-142-3p and HMGB1 expression. *P<.05. Translational Oncology 2017 10, 410-418DOI: (10.1016/j.tranon.2017.03.003) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Expression levels of miR-142-3p, HMGB1, P-gp, and autophagy-related proteins in human drug-resistant AML cell lines HL-60/ATRA and HL-60/ADR and their parental cell line HL-60. The expressions of miR-142-3p (A) and HMGB1 mRNA (B) in the human bone marrow stromal cell line HS-5 cells, AML cell line HL-60, and drug-resistant AML cell lines HL-60/ATRA and HL-60/ADR were evaluated by qRT-PCR. The levels of HMGB1 (C and D), P-gp (E and F), and autophagy-related proteins (Atg5, LC3-I, and LC3-II) (G and H) in HS-5 cells, AML cell line HL-60, and drug-resistant AML cell lines HL-60/ATRA and HL-60/ADR were detected by Western blot. *P<.05. Translational Oncology 2017 10, 410-418DOI: (10.1016/j.tranon.2017.03.003) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Effect of miR-142-3p on drug sensitivity of resistant AML cells. (A) Cell survival rates were detected by MTT assay in HL-60 and HL-60/ATRA treated with various concentrations (0.5, 1, 2, 4, and 8 μM) of ATRA for 72 hours. (B) Cell survival rates were detected by MTT assay in HL-60 and HL-60/ADR incubated with various concentrations of ADR (50, 100, 200, 300, and 400 nM) for 72 hours. Cell viability in HL-60/ATRA (C) and HL-60/ADR (D) cells transfected with miR-142-3p or miR-control or along with 4 μM ATRA or 300 nM ADR was evaluated by MTT assay. Apoptosis in HL-60/ATRA (E) and HL-60/ADR (F) cells transfected with miR-142-3p or miR-control or along with 4 μM ATRA (E) or 300 nM ADR (F) was assessed by flow cytometry. *P<.05. Translational Oncology 2017 10, 410-418DOI: (10.1016/j.tranon.2017.03.003) Copyright © 2017 The Authors Terms and Conditions

Figure 4 HMGB1 reversed miR-142-3p–induced drug sensitivity in HL-60/ATRA and HL-60/ADR cells. (A and B) After cells treated with ATRA or ADR were transfected with miR-142-3p, miR-control or along with pcDNA-HMGB1 or vector, cell viability was determined by MTT assay. (C and D) After cells treated with ATRA or ADR were transfected with miR-142-3p, miR-control or along with pcDNA-HMGB1 or vector, apoptosis was examined by flow cytometry. *P<.05. Translational Oncology 2017 10, 410-418DOI: (10.1016/j.tranon.2017.03.003) Copyright © 2017 The Authors Terms and Conditions

Figure 5 miR-142-3p directly targeted HMGB1 and negatively regulated its expression. (A) Diagram of putative miR-142-3p binding sequence in HMGB1 3′UTR and its mutant in luciferase reporter assay. (B) Luciferase reporter assay was performed to measure luciferase activity in HL-60/ATRA or HL-60/ADR cells cotransfected with luciferase reporter vectors (pGL3-HMGB1-3′UTR-WT or pGL3-HMGB1-3′UTR-MUT) with miR-142-3p or miR-control. (C) qRT-PCR was carried out to assess the expression of HMGB1 mRNA in HL-60/ATRA and HL-60/ADR cells transfected with miR-142-3p, anti–miR-142-3p, or respective controls. Western blot was used to determine the level of HMGB1 in HL-60/ATRA (D) and HL-60/ADR (E) cells transfected with miR-142-3p, anti–miR-142-3p, or respective controls. *P<.05. Translational Oncology 2017 10, 410-418DOI: (10.1016/j.tranon.2017.03.003) Copyright © 2017 The Authors Terms and Conditions

Figure 6 miR-142-3p upregulation enhanced drug sensitivity of AML cells through reducing P-gp and suppressing autophagy by targeting HMGB1. The level of P-gp in HL-60/ATRA (A) and HL-60/ADR (B) cells transfected with miR-142-3p or miR-control, or together with pcDNA-HMGB1 or vector were detected by Western blot. The levels of autophagy related proteins Atg5, LC3-I, and LC3-II were analyzed by Western blot in HL-60/ATRA (C) and HL-60/ADR (D) cells transfected with miR-142-3p or miR-control, or together with pcDNA-HMGB1 or vector. *P<.05. Translational Oncology 2017 10, 410-418DOI: (10.1016/j.tranon.2017.03.003) Copyright © 2017 The Authors Terms and Conditions