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. 2024 Jun 24;19(1):49.
doi: 10.1186/s13062-024-00490-1.

Silencing LINC00987 ameliorates adriamycin resistance of acute myeloid leukemia via miR-4458/HMGA2 axis

Yue Liu #  1 , Xiao-Ya Zhu #  2 , Li-Li Liao  3 , Zhan-Hui Zhang  3 , Tao-Sheng Huang  3 , Ling Zhang  4 , Xi-Wen Jiang  5 , Yi Ma  6   7   8
Affiliations

Silencing LINC00987 ameliorates adriamycin resistance of acute myeloid leukemia via miR-4458/HMGA2 axis

Yue Liu et al. Biol Direct. .

Abstract

Background: Most patients with acute myeloid leukemia (AML) eventually develop drug resistance, leading to a poor prognosis. Dysregulated long gene non coding RNAs (lincRNAs) have been implicated in chemoresistance in AML. Unfortunately, the effects of lincRNAs which participate in regulating the Adriamycin (ADR) resistance in AML cells remain unclear. Thus, the purpose of this study is to determine LINC00987 function in ADR-resistant AML.

Methods: In this study, ADR-resistant cells were constructed. LINC00987, miRNAs, and HMGA2 mRNA expression were measured by qRT-PCR. P-GP, BCRP, and HMGA2 protein were measured by Western blot. The proliferation was analyzed by MTS and calculated IC50. Soft agar colony formation assay and TUNEL staining were used to analyze cell colony formation and apoptosis. Xenograft tumor experiment was used to analyze the xenograft tumor growth of ADR-resistant AML.

Results: We found that higher expression of LINC00987 was observed in AML patients and associated with poor overall survival in AML patients. LINC00987 expression was increased in ADR-resistant AML cells, including ADR/MOLM13 and ADR/HL-60 cells. LINC00987 downregulation reduces ADR resistance in ADR/MOLM13 and ADR/HL-60 cells in vitro and in vivo, while LINC00987 overexpression enhanced ADR resistance in MOLM13 and HL-60 cells. Additionally, LINC00987 functions as a competing endogenous RNA for miR-4458 to affect ADR resistance in ADR/MOLM13 and ADR/HL-60 cells. HMGA2 is a target of miR-4458. LINC00987 knockdown and miR-4458 overexpression reduced HMGA2 expression. HMGA2 overexpression enhanced ADR resistance, which reversed the function of LINC00987 silencing in suppressing ADR resistance of ADR/MOLM13 and ADR/HL-60 cells.

Conclusions: Downregulation of LINC00987 weakens ADR resistance by releasing miR-4458 to deplete HMGA2 in ADR/MOLM13 and ADR/HL-60. Therefore, LINC00987 may act as the therapeutic target for treating chemoresistant AML.

Keywords: Acute myeloid leukemia; Adriamycin; Long non-coding RNA; microRNA.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
LINC00987 expression was higher in AML patients and associated with poor overall survival. (A) The relationship between LINC00987, LINC00152, and LINC00982 expression with LAML survival were analyzed using GEPIA. (B) LINC00987, LINC00152, and LINC00982 expression in LAML patients were analyzed via GEPIA. (C) LINC00987, LINC00152, and LINC00982 expression in MOLM13, HL-60, ADR/MOLM13, and ADR/HL-60 cells were measured via RT-qPCR. ***P < 0.001
Fig. 2
Fig. 2
Decreased expression of LINC00987 reduces the cell viability and ADR-resistance of AML cells. (A) LINC00987 expression in ADR/MOLM13 and ADR/HL-60 cells was measured via RT-qPCR after siRNA transfection at 24 h. (B) The proliferation of ADR/MOLM13 and ADR/HL-60 cells was detected by CCK8 after siRNA transfection at 24 h under 0.2 µM ADR treatment. The cell viability was calculated according to proliferation. (C) Clone formation of ADR/MOLM13 and ADR/HL-60 cells was detected by soft agar colony formation assay. The number of cell clone formations was counted by imaging at 100x magnification. (D) Xenograft tumor was used to analyze the effect of LINC00987-silenced on the size of tumor growth. And LINC00987 expression in Xenograft tumor was measured via RT-qPCR. *P < 0.005 and ***P < 0.001
Fig. 3
Fig. 3
LINC00987 downexpression reduces the ADR resistance of ADR-resistant AML cells under 0.2 µM ADR treatment. (A) The IC50 of ADR for si1-LINC00987-transfected ADR/MOLM13 and ADR/HL-60 cells were shown after different concentrations of ADR treatment at 24 h. (B) Under 0.2 µM ADR treatment, P-GP and BCRP expression in ADR/MOLM13 and ADR/HL-60 cells were measured by western blotting. (C) Apoptosis of ADR/MOLM13 and ADR/HL-60 cells were detected by TUNEL staining and imaging at 400× magnification. ***P < 0.001
Fig. 4
Fig. 4
LINC00987 overexpression enhanced the cell viability, clone formation and ADR-resistance of AML cells. (A) LINC00987 expression in MOLM13 and HL-60 cells were measured by RT-qPCR after transfection with pcDNA-LINC00987 and pcDNA-Empty at 24 h. (B) The proliferation of MOLM13 and HL-60 cells was detected by CCK8 after transfection with pcDNA-LINC00987 and pcDNA-Empty at 24 h under 0.2 µM ADR treatment. Then, the cell viability was calculated according to proliferation. (C) Clone formation was detected by soft agar assay colony formation. The number of cell clone formations was counted by imaging at 100x magnification. (D) The IC50 of ADR for the pcDNA-LINC00987-transfected MOLM13 and HL-60 cells were shown after different concentrations of ADR treatment at 24 h. (E) Under 0.2 µM ADR treatment, P-GP and BCRP expression in MOLM13 and HL-60 cells were measured by Western blot. (F) Apoptosis was detected by TUNEL staining and imaging at 400x magnification. ***P < 0.001
Fig. 5
Fig. 5
miR-4458 regulates cell viability and ADR-resistance to affect LINC00987 function in ADR-resistant AML cells. (A) miR-4458 expression in ADR/MOLM13 and ADR/HL-60 cells were measured via RT-qPCR after co-transfection at 24 h. (B) The proliferation of ADR/MOLM13 and ADR/HL-60 cells was detected by CCK8 after co-transfection at 24 h under 0.2 µM ADR treatment. Then, the cell viability was calculated according to proliferation. (C) Clone formation of ADR/MOLM13 and ADR/HL-60 cells was detected by soft agar colony formation assay after co-transfection at 24 h under 0.2 µM ADR treatment. The number of cell clone formations was counted by imaging at 100× magnification. (D) The IC50 of ADR for co-transfected ADR/MOLM13 and ADR/HL-60 cells were shown after different concentrations of ADR treatment at 24 h. (E) Under 0.2 µM ADR treatment, P-GP and BCRP expression in ADR/MOLM13 and ADR/HL-60 cells were measured by western blotting after si1-LINC00987 and miR-4458 inhibitor co-transfection. ***P < 0.001
Fig. 6
Fig. 6
HMGA2 promotes cell viability and ADR-resistance to assist in LINC00987 function in ADR-resistant AML cells. (A) HMGA2 protein expression in ADR/MOLM13 and ADR/HL-60 cells were measured by Western blot after si1-LINC00987 and pcDNA-HMGA2 co-transfection at 24 h. (B) The proliferation of ADR/MOLM13 and ADR/HL-60 cells was detected using CCK8 after co-transfection at 24 h under 0.2 µM ADR treatment. Then, the cell viability was calculated according to proliferation. (C) Clone formation of ADR/MOLM13 and ADR/HL-60 cells was detected by soft agar colony formation assay after co-transfection at 24 h under 0.2 µM ADR treatment. The number of cell clone formations was counted by imaging at 100× magnification. (D) The IC50 of ADR for co-transfected ADR/MOLM13 and ADR/HL-60 cells were shown after different concentrations of ADR treatment at 24 h. (E) Under 0.2 µM ADR treatment, P-GP and BCRP expression in ADR/MOLM13 and ADR/HL-60 cells were measured by western blotting after si1-LINC00987 and pcDNA-HMGA2 co-transfection. ***P < 0.001
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