ACSM3 suppresses proliferation and induces apoptosis and cell cycle arrest in acute myeloid leukemia cells via the regulation of IGF2BP2

Abstract

Acyl-CoA medium-chain synthetase-3 (ACSM3) has been reported to be involved in the malignant progression of multiple types of human cancer. Nevertheless, the role of ACSM3 in acute myeloid leukemia (AML) and its exact mechanism of action are as yet undefined. In the present study, the expression levels of ACSM3 and IGF2 mRNA-binding protein 2 (IGF2BP2) were evaluated using the Gene Expression Profiling Interactive Analysis database and AML cells. The Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were employed for the estimation of the cell proliferative activity. Induction of apoptosis and the assessment of the cell cycle were measured using flow cytometry and western blotting, respectively. The interaction of ACSM3 with IGF2BP2 was confirmed using an RNA immunoprecipitation assay. mRNA stabilization of ACSM3 following actinomycin D treatment was evaluated using reverse transcription-quantitative PCR analysis. The data indicated that the expression levels of ACSM3 were significantly downregulated, whereas those of IGF2BP2 were upregulated in tissues and AML cells. Downregulation of ACSM3 expression was closely associated with poor overall survival of patients with AML. ACSM3 overexpression repressed cell proliferative activity and induced apoptosis and cell cycle arrest. IGF2BP2 downregulated ACSM3 expression by reducing the stability of ACSM3 mRNA. In addition, IGF2BP2 overexpression counteracted the effects of ACSM3 overexpression noted on proliferation, induction of apoptosis and cell cycle arrest of HL-60 cells. In conclusion, ACSM3 repressed the cell proliferative activity and facilitated induction of apoptosis and cell cycle arrest in AML cells by modulating the expression of IGF2BP2.

Keywords: IGF2 mRNA binding protein 2; RNA-binding protein; acute myeloid leukemia; acyl-CoA medium-chain synthetase-3; apoptosis; cycle arrest.

Figure 1

ACSM3 is downregulated in AML and is associated with poor prognosis. (A) Analysis of ACSM3 expression in multiple types of cancer by GEPIA database. (B) Analysis of ACSM3 expression in patients with AML and healthy controls by GEPIA database. (C) Association of ACSM3 and overall survival in patients with AML, by GEPIA database. (D) mRNA expression and (E) protein expression of ACSM31 in AML cells detected using reverse transcription-quantitative PCR and western blotting, respectively. ^*P<0.05. ^***P<0.001 vs. HS-5. ACSM3, acyl-CoA medium-chain synthetase-3; GEPIA, gene expression profiling interactive analysis; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; AML/LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma.

Figure 2

ACSM3 overexpression inhibits HL-60 cell proliferation. (A) mRNA and (B) protein expression levels of ACSM3 in transfected acute myeloid leukemia cells were detected by reverse transcription-quantitative PCR and western blotting. Cell proliferation was evaluated by (C) Cell Counting Kit-8 assay and (D) EdU staining. Results are displayed as the mean ± SD. ^***P<0.001 vs. Ov-NC. ACSM3, acyl-CoA medium-chain synthetase-3; EdU, 5-ethynyl-2'-deoxyuridine; NC, negative control; OD, optical density; Ov, overexpressing.

Figure 3

Upregulated ACSM3 promotes apoptosis and cycle arrest of HL-60 cells. (A) Apoptosis detected and (B) quantified using TUNEL assay. (C) Western blotting used to assess the protein levels of Bcl-2, Bax and cleaved caspase 3/caspase 3. (D) Cell cycle was detected and (E) quantified using flow cytometry analysis. (F) Western blotting used to evaluate the protein levels of CDK4, CDK6 and Cyclin D1. Results are the mean ± SD. ^**P<0.01 and ^***P<0.001 vs. Ov-NC. ACSM3, acyl-CoA medium-chain synthetase-3; NC, negative control; Ov, overexpressing. Bcl-2, B-cell lymphoma-2; Bax, Bcl2-Associated X; CDK4, cyclin dependent kinase 4; CDK6, cyclin dependent kinase 6.

Figure 4

IGF2BP2 reduces the stability of ACSM3 mRNA and downregulates ACSM3 expression. (A) Analysis of IGF2BP2 expression in patients with AML and healthy controls by GEPIA database. (B) mRNA and (C) protein expression levels of IGF2BP2 in HS-5 and HL-60 cells were detected by RT-qPCR and western blotting, respectively. (D) RNA immunoprecipitation assay confirmed the binding of IGF2BP2 and ACSM3 mRNA. (E) mRNA and (F) protein expression levels of IGF2BP2 in HL-60 cells transfected with Ov-IGF2BP2 were detected by RT-qPCR and western blotting, respectively. (G) RNA stability assay was performed to assess the stability of ACSM3 mRNA. (H) mRNA and (I) protein expression levels of ACSM3 in HL-60 cells transfected with Ov-IGF2BP2 were detected using RT-qPCR and western blotting, respectively. ^*P<0.05 compared to healthy controls. ^***P<0.001 vs. HS-5, IgG and Ov-NC. ACSM3, acyl-CoA medium-chain synthetase-3; AML/LAML, acute myeloid leukemia; IGFBP2, IGF2 binding protein 2; NC, negative control; Ov, overexpressing; RT-qPCR, reverse transcription-quantitative PCR.

Figure 5

Effects of ACSM3 on the proliferation, apoptosis and cell cycle of HL-60 cells is associated with regulation of IGF2BP2. Cell proliferation was evaluated using (A) CCK-8 assay and (B) EdU staining. (C) Apoptosis was detected and (D) quantified using TUNEL assay. (E) Western blotting was used to assess the protein levels of Bcl-2, Bax and cleaved caspase 3/caspase 3. (F) Cell cycle was detected and (G) quantified using flow cytometry analysis. (H) Western blotting was used to evaluate the protein levels of CDK4, CDK6 and Cyclin D1. Results are displayed as the mean ± SD. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. Control. ^#P<0.05 and ^###P<0.001 vs. Ov-ACSM3 + Ov-NC. ACSM3, acyl-CoA medium-chain synthetase-3; EdU, 5-ethynyl-2'-deoxyuridine; Ov, overexpressing; NC, negative control; OD, optical density. Bcl-2, B-cell lymphoma-2; Bax, Bcl2-Associated X; CDK4, cyclin dependent kinase 4; CDK6, cyclin dependent kinase 6.