microRNA-628 inhibits the proliferation of acute myeloid leukemia cells by directly targeting IGF-1R

Abstract

Background: A variety of microRNAs (miRNAs) are aberrantly expressed in acute myeloid leukemia (AML), and these dysregulated miRNAs perform crucial roles in tumorigenesis and progression of AML. miR-628-3p (miR-628), one of the miRNAs dysregulated in multiple types of human cancers, exerts antitumor roles in different cancer types. However, no specific study has explored the expression pattern and role of miR-628 in AML.

Materials and methods: In this study, RT-qPCR was performed to detect miR-628 expression in AML tissues and cell lines. CCK-8 assay, flow cytometry analysis and xenograft tumor experiment was carried out to determine the functions of miR-628 in AML cells. The possible mechanism underlying the activity of miR-628 in AML cells was also explored using a series of experiments.

Results: Our results revealed the downregulated expression of miR-628 in patients with AML and AML cell lines. Ectopic expression of miR-628 resulted in the inhibition of AML cell proliferation and induction of cell cycle arrest and apoptosis in vitro and attenuation of tumor growth in vivo. Insulin-like growth factor 1 receptor (IGF-1R) was identified as a direct target gene of miR-628 in AML cells. IGF-1R expression was upregulated in patients with AML and upregulation of IGF-1R expression inversely correlated with miR-628 level. Furthermore, IGF-1R knockdown imitated the tumor suppressive effect of miR-628 in AML cells. Restoration of IGF-1R expression abrogated the effects of miR-628 on the proliferation, cycle status, and apoptosis rate of AML cells. miR-628 inhibited the activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) pathway in AML cells both in vitro and in vivo through the inhibition of IGF-1R expression.

Conclusion: Our results demonstrate that miR-628 exhibits antitumor effects in AML through the direct targeting of IGF-1R and regulation of PI3K/Akt pathway, suggestive of its potential role as a therapeutic target in patients with this aggressive hematological malignant tumor.

Keywords: PI3K/Akt pathway; acute myeloid leukemia; apoptosis; cell cycle; insulin-like growth factor 1 receptor; microRNA-628; proliferation.

Figure 1

miR-628 expression is downregulated in AML. Notes: (A) RT-qPCR analysis was performed to detect miR-628 expression in the bone marrow samples derived from 29 patients with AML and 23 healthy controls. *P<0.05 vs healthy controls. (B) The expression status of miR-628 in three human AML cell lines (HL-60, Kasumi-1, and THP-1) and a normal bone marrow cell line (HS-5) was determined with RT-qPCR analysis. *P<0.05 vs HS-5. Abbreviations: AML, acute myeloid leukemia; RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Figure 2

miR-628 overexpression inhibits proliferation, induces cell cycle arrest, and promotes apoptosis of HL-60 and THP-1 cells. Notes: (A) RT-qPCR analysis was performed to measure miR-628 expression in HL-60 and THP-1 cells after transfection with miR-628 mimics or miR-NC. *P<0.05 vs miR-NC. (B) CCK-8 assay was used to evaluate the proliferative ability of miR-628-overexpressing HL-60 and THP-1 cells. *P<0.05 vs miR-NC. (C, D) Cell cycle status and apoptotic rate of HL-60 and THP-1 cells treated with miR-628 mimics or miR-NC were determined using flow cytometry analysis. *P<0.05 vs miR-NC. Abbreviations: CCK-8, cell counting kit-8; miR-NC, miRNA mimics negative control; RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Figure 3

miR-628 directly targets IGF-1R in AML cells. Notes: (A) The putative wild-type (WT) and mutated (MUT) binding sites for miR-628 in the 3′-UTR of IGF-1R are shown. (B) miR-628 mimics or miR-NC and a luciferase plasmid carrying the WT or MUT miR-628-binding site were transfected into HL-60 and THP-1 cells. After 48 hours of transfection, the transfected cells were harvested and subjected to quantification of luciferase activity using a Dual-Luciferase Reporter Assay System. *P<0.05 vs miR-NC. (C) The expression levels of IGF-1R mRNA in the bone marrow samples were derived from 29 patients with AML and 23 healthy controls were detected with RT-qPCR. *P<0.05 vs miR-NC. (D) Spearman’s correlation analysis was used to examine the correlation between miR-628 and IGF-1R mRNA levels in patients with AML. r=-0.5393, P=0.0025. (E, F) RT-qPCR and Western blot analysis were performed to detect the expression levels of IGF-1R mRNA and protein, respectively, in miR-628-overexpressing HL-60 and THP-1 cells. Western blot analysis was repeated at least three times. *P<0.05 vs miR-NC. Abbreviations: AML, acute myeloid leukemia; IGF-1R, insulin-like growth factor 1 receptor; miR-NC, miRNA mimics negative control; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; 3′-UTR, 3′-untranslated regions.

Figure 4

Silencing of IGF-1R expression simulates the effects of miR-628 overexpression in HL-60 and THP-1 cells. Notes: (A) Western blot analysis was carried out to determine IGF-1R expression in HL-60 and THP-1 cells treated with IGF-1R siRNA or NC siRNA. Western blot analysis was repeated at least three times. *P<0.05 vs NC siRNA. (B) The effect of IGF-1R inhibition on HL-60 and THP-1 cell proliferation was determined by CCK-8 assay. *P<0.05 vs NC siRNA. (C, D) Flow cytometry analysis was used to assess the cell cycle status and apoptotic rate of HL-60 and THP-1 cells after transfection with IGF-1R siRNA or NC siRNA. *P<0.05 vs NC siRNA. Abbreviations: CCK-8, cell counting kit-8; IGF-1R, insulin-like growth factor 1 receptor; NC, negative control; siRNA, small-interfering RNA.

Figure 5

IGF-1R rescues the miR-628-induced proliferation, migration, and invasion of HL-60 and THP-1 cells. Notes: HL-60 and THP-1 cells were cotransfected with miR-628 mimics and pc-IGF-1R or pcDNA3.1. (A) After 72 hours of transfection, Western blot analysis was performed to detect IGF-1R protein expression. Western blot analysis was repeated at least three times. *P<0.05 vs miR-NC. **P<0.05 vs miR-628 mimics + pcDNA3.1. (B–D) The proliferation, cell cycle status, and apoptotic rate of HL-60 and THP-1 cells treated as above were evaluated using CCK-8 assay and flow cytometry analysis. *P<0.05 vs miR-NC. **P<0.05 vs miR-628 mimics + pcDNA3.1. Abbreviations: CCK-8, cell counting kit-8; IGF-1R, insulin-like growth factor 1 receptor; miR-NC, miRNA mimics negative control.

Figure 6

miR-628 targets IGF-1R to inhibit the activation of PI3K/Akt signaling pathway in AML cells. Notes: miR-628 mimics and pc-IGF-1R or pcDNA3.1 were introduced into HL-60 and THP-1 cells. Following 72 hours of incubation, the expression levels of molecules related to the PI3K/Akt pathway were quantified with Western blot analysis. Western blot analysis was repeated at least three times. *P<0.05 vs miR-NC. **P<0.05 vs miR-628 mimics + pcDNA3.1. Abbreviations: AML, acute myeloid leukemia; IGF-1R, insulin-like growth factor 1 receptor; miR-NC, miRNA mimics negative control; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase.

Figure 7

miR-628 inhibits the growth of AML tumors in vivo. Notes: (A) Representative images of the xenograft tumors obtained from miR-628 mimics- or miR-NC-transfected cells. (B) The tumor volume was detected every 2 days for 4 weeks. The tumor volumes were determined with the following formula: Volume (mm³) = width (mm²) × length (mm)/2. *P<0.05 vs miR-NC. (C) The xenograft tumors formed were excised after 4 weeks. The weights were significantly lower in the xenografts obtained from miR-628 group than those obtained from the miR-NC group. *P<0.05 vs miR-NC. (D) The expression level of miR-628 in the tumor xenografts was detected with RT-qPCR. *P<0.05 vs miR-NC. (E) Western blot analysis was used to measure the expression levels of IGF-1R, p-PI3K, PI3K, p-Akt, and Akt proteins in the tumor xenografts. Western blot analysis was repeated at least three times. *P<0.05 vs miR-NC. Abbreviations: AML, acute myeloid leukemia; IGF-1R, insulin-like growth factor 1 receptor; miR-NC, miRNA mimics negative control; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; RT-qPCR, reverse-transcription quantitative polymerase chain reaction.