Yonsei Med J.  2019 Oct;60(10):924-934. 10.3349/ymj.2019.60.10.924.

microRNA-146a Promotes Growth of Acute Leukemia Cells by Downregulating Ciliary Neurotrophic Factor Receptor and Activating JAK2/STAT3 Signaling

• Wang L
• Zhang H
• Lei D

• KMID: 2459145
• DOI: http://doi.org/10.3349/ymj.2019.60.10.924

Abstract

PURPOSE
Acute leukemia (AL) is classified as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This study aimed to investigate the effect of miR-146a on childhood AL and its underlying molecular mechanisms.
MATERIALS AND METHODS
Bone marrow samples were obtained from 39 AL children and 10 non-cancer controls. The expressions of miR-146a and ciliary neurotrophic factor receptor (CNTFR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in ALL and AML pediatric patients, as well as ALL (Jurkat) and AML (HL-60) cells. Correlations between miR-146a and clinical indicators were explored. A targeting relationship between miR-146a and CNTFR was detected by dual luciferase reporter gene assay. Cell proliferation, apoptosis, migration, and invasion of Jurkat and HL-60 cells were measured by MTT assay, flow cytometry, and transwell assay, respectively. LIF expression was detected by qRT-PCR in Jurkat and HL-60 cells. The expression of p-JAK2, JAK2, p-STAT3, and STAT3 in HL-60 cells was measured by Western blot.
RESULTS
miR-146a was increased in ALL and AML pediatric patients, while CNTFR was decreased. miR-146a expression was associated with immunophenotype, karyotype, fusion gene, and SIL-TAL1. CNTFR was a target gene of miR-146a. miR-146a could promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis in Jurkat and HL-60 cells by downregulating CNTFR. Meanwhile, miR-146a inhibited the expression of LIF and activated JAK2/STAT3 pathway by downregulating CNTFR.
CONCLUSION
miR-146a could promote the proliferation, migration, and invasion and inhibit the apoptosis of AL Jurkat and HL-60 cells by downregulating CNTFR and activating the JAK2/STAT3 pathway.

Keyword

miR-146a; acute leukemia; CNTFR; JAK/STAT pathway

MeSH Terms

Apoptosis
Blotting, Western
Bone Marrow
Cell Proliferation
Child
Ciliary Neurotrophic Factor*
Flow Cytometry
Genes, Reporter
HL-60 Cells
Humans
Karyotype
Leukemia*
Leukemia, Myeloid, Acute
Luciferases
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Real-Time Polymerase Chain Reaction
Receptor, Ciliary Neurotrophic Factor*
Ciliary Neurotrophic Factor
Luciferases
Receptor, Ciliary Neurotrophic Factor

Figure

• 

  Fig. 1 The expression of miR-146a in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) pediatric patients. Data are presented as a mean±standard deviation. Experiments were performed three times. *p<0.05, vs. Control group; †p<0.05, vs. AML group.

• 

  Fig. 2 Ciliary neurotrophic factor receptor (CNTFR) is a target of miR-146a. (A) The expression of CNTFR was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) pediatric patients. (B) The binding target of CNTFR and miR-146a was predicted by TargetScan. (C) Luciferase activity was measured by dual luciferase reporter gene assay. (D) The correlation of leukemic inhibitory factor (LIF) and CNTFR in ALL and AML pediatric patients. Data are presented as a mean±standard deviation. Experiments were performed three times. *p<0.05, vs. Control group and wild-type (WT)+negative control (NC) group. MT, mutant-type.

• 

  Fig. 3 miR-146a promotes the proliferation and suppresses the apoptosis of Jurkat and HL-60 cells. (A) miR-146a expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in Jurkat and HL-60 cells. (B) Proliferation of Jurkat and HL-60 cells measured by MTT assay. (C) Apoptosis of Jurkat and HL-60 cells detected by flow cytometry. Data are presented as a mean±standard deviation. Experiments were performed three times. *p<0.05, vs. Mock and mimics-negative control (NC) group. OD, optical density.

• 

  Fig. 4 miR-146a promotes the proliferation and suppresses the apoptosis of Jurkat and HL-60 cells by downregulating ciliary neurotrophic factor receptor (CNTFR). (A) Proliferation of Jurkat and HL-60 cells measured by MTT assay. (B) Apoptosis of Jurkat and HL-60 cells detected by flow cytometry. (C) Quantitative data of cell apoptosis ratio in Jurkat and HL-60 cells. Data are presented as a mean±standard deviation. Experiments were performed three times. *p<0.05, vs. anti-miR-NC+siNC; †p<0.05, vs. anti-miR-146a+siNC; ‡p<0.05, vs. anti-miR-NC+siCNTFR. NC, negative control; OD, optical density.

• 

  Fig. 5 miR-146a increases the migration and invasion of Jurkat and HL-60 cells by downregulating ciliary neurotrophic factor receptor (CNTFR). Cell migration (A) and invasion (B) of Jurkat and HL-60 cells detected by transwell assay after crystal violet staining. Data are presented as a mean±standard deviation. Experiments were performed three times. Original magnification: ×100. *p<0.05, vs. anti-miR-NC+siNC; †p<0.05, vs. anti-miR-146a+siNC; ‡p<0.05, vs. anti-miR-NC+siCNTFR. NC, negative control.

• 

  Fig. 6 miR-146a inhibits the expression of leukemic inhibitory factor (LIF) in Jurkat and HL-60 cells by downregulating ciliary neurotrophic factor receptor (CNTFR). (A) The expression of LIF in Jurkat cells detected by quantitative real-time polymerase chain reaction (qRT-PCR). (B) The expression of LIF in HL-60 cells detected by qRT-PCR. Data are presented as a mean±standard deviation. Experiments were performed three times. *p<0.05, vs. anti-miR-NC+siNC; †p<0.05, vs. anti-miR-146a+siNC; ‡p<0.05, vs. anti-miR-NC+siCNTFR.

• 

  Fig. 7 miR-146a activates the JAK2/STAT3 pathway by downregulating ciliary neurotrophic factor receptor (CNTFR) in Jurkat and HL-60 cells. (A) The expression of p-JAK2, JAK2, p-STAT3 and STAT3 in Jurkat cells detected by western blot. (B) The expression of p-JAK2, JAK2, p-STAT3 and STAT3 in HL-60 cells detected by western blot. Data are presented as a mean±standard deviation. Experiments were performed three times. *p<0.05, vs. anti-miR-NC+siNC; †p<0.05, vs. anti-miR-146a+siNC; ‡p<0.05, vs. anti-miR-NC+siCNTFR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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