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Introduction

MicroRNAs (miRs) are a class of small non-coding RNAs that regulate gene expression by targeting messenger (m)RNA. A previous study suggested that an estimated third of all genes are regulated by miRs (1). miRs usually bind to the 3′-untranslated region (UTR) of mRNA sequences with imperfect complementarity. Consensus sequences of miR are also located in 5′-UTRs, however their regulatory roles differ (2,3). Numerous miRs have been identified to date. A previous study demonstrated that miR participated in cellular differentiation, proliferation and survival in various tissue types. In addition, aberrant miR expression was identified in a number of different pathologies (4). The specific role of miRs has been identified in numerous tumor types, including suppressor or oncogenic functions (5). However, few studies have focused on the role of miRs in acute myeloid leukemia (AML). In chronic lymphocytic leukemia, miRs have been suggested to possess important roles (6,7). Furthermore, miR-223 and -155 have been reported to be involved in the pathogenesis of AML (8,9). These studies suggested that miRs may be involved in myeloid transformation, which leads to an increase in genomic alterations that result in consecutive transformation events in the leukemic clones during leukemic development (10). At present, abnormal signaling molecules, such as abnormal growth factor receptors and transcription factors, have been identified in specific leukemic phenotypes and treatment strategies (11).

In addition to miRs, AU-rich element (ARE)-mediated transcript degradation is significant in gene regulation at the post-transcriptional level (12). Numerous cancer-associated transcripts contain AREs in the 5′ or 3′-UTR, including cytokines, growth factors and invasion factors, and regulate growth and proliferation (13). Alterations to the stability and translation efficiency of mRNAs result in the disruption of gene expression patterns (13). Human antigen R (HuR) is a human embryonic lethal abnormal vision-like (ELAV) RNA-binding protein, which belongs to the Hu family (14). In mammalian cells, HuR binds to AREs to adjust the instability of mRNAs (15). To regulate protein translation, HuR recognizes and binds to AREs in the 3′-UTR of mRNAs. Furthermore, HuR was reported to increase the stability of the majority of these mRNAs (16–18). A previous study demonstrated that HuR localizes primarily to the nucleus, and was able to move between the nucleus and cytoplasm. This trans-location enables HuR to efficiently stabilize the mRNAs (19). Increasing evidence has suggested that HuR is important in carcinogenesis and cancer progression by regulating the expression of numerous target genes, such as p53, p21, p27, and B cell lymphoma 2 (Bcl-2) (20).

A previous study reported that miR-519 may regulate the protein expression levels of HuR by binding to specific regions in the 3′ or 5′-UTR (21). In the present study, the main aim was to examine the role of miR-519 in AML. Furthermore, the biological role of HuR in AML cell proliferation and migration was investigated.

Materials and methods

Cell culture and human tissue samples

The HL60 human AML cell line [American Type Culture Collection (ATCC), Manassas, VA, USA] was cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies). The NOMO1 human myeloid cell line (ATCC) was cultured in RPMI-1640 (Invitrogen Life Technologies) supplemented with 10% FBS.

The bone marrow tissue samples from 60 patients (29 males and 31 females with an average age of 32±4.4 years) with newly diagnosed pediatric AML and 60 patients (30 males and 30 females with an average age of 36±4.3 years) with healthy pediatric bone marrow were collected between January 2013 and January 2014. The leukemia diagnoses were made according to standard morphological criteria based on immunohistochemistry, immunophenotyping and cytogenetic studies, according to the AIEOP-2002 AML pediatric protocol (22). Informed written consent in compliance with the Helsinki protocol was obtained from the patients. The study was approved by the ethics committee of The Third Hospital of Chinese People's Liberation Army (Baoji, China).

Transient transfection

Prior to transfection, the cells were seeded in 6-well plates at a density of 1×105 cells/well with 2 ml DMEM, containing 10% FBS, and 100 U/ml penicillin and 100 U/ml streptomycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). miR-519 mimic, miR-519 inhibitor or negative control (Shanghai GenePharma Co., Ltd., Shanghai, China) were pre-incubated with HiPerFect transfection reagent (Qiagen China Co., Ltd., Shanghai, China) at room temperature for 10 min. Briefly, 12 µl Hiperfect transfection reagent was incubated with 100 µl L-DMEM without serum for 5 min. Meanwhile, miR-519 mimics, inhibitor or negative control was incubated with L-DMEM without serum for 5 min. Subsequently, they were mixed together for 10 min. The complex was then transfected into the HL60 cells at a final concentration of 50 nM. The transfected cells were incubated in RPMI-1640 supplemented with 10% FBS for a further 48 h.

RNA extraction and miR quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

The RNA from the cell lines and from the bone marrow tissue samples was isolated following homogenization using a mirVana miRNA Isolation kit (Ambion Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA), which was then used to conduct the RT-qPCR. Taqman® MicroRNA Reverse Transcription kit (Applied Biosystems Life Technologies, Foster City, CA, USA) were conducted using the stem-loop method to detect the expression levels of mature miRs (23). Total RNA (10 ng) was reverse transcribed and mixed with specific stem-loop primers (Applied Biosystems Life Technologies). All RT-qPCRs were conducted in triplicate and the miR expression levels relative to U6 small nuclear (sn)RNA (RNU6B) were calculated using the comparative Ct (ΔΔCt) method (24).

Protein extraction, western blotting and antibodies

Proteins were extracted from the HL60 cells using radio-immunoprecipitation assay buffer containing 1% Triton X-100, 150 mmol/l NaCl, 5 mmol/l EDTA and 10 mmol/l Tris-HCl (pH 7.0; Beijing Solarbio Science & Technology Co., Ltd.) supplemented with a protease inhibitor cocktail (cat. no. P4830; Sigma-Aldrich, St. Louis, MO, USA). The cell lysates were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Qiagen China Co., Ltd.). Following blocking with 8% milk in phosphate-buffered saline (PBS; pH 7.5; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China), the membranes were incubated with the following primary antibodies: Anti-HuR (cat. no. 12582; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:1,000), anti-Bcl-2 (cat. no. 2876; Cell Signaling Technology, Inc.; 1:1,000), anti-Bcl-2-like protein 4 (Bax; cat. no. 2772; Cell Signaling Technology, Inc.; 1:1,000), and anti-β-actin (cat. no. H-190; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:3,000). Following overnight incubation at 4°C, the membranes were incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Ig) G secondary antibodies (all at a 1:5,000; cat. no. ZB-2301; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), and immunodetection was achieved using the Electrochemiluminescence Plus Detection system (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. β-actin served as an internal control.

MTT assay

To investigate the effects of miR-519 on cell viability, 5×103 cells/well were seeded in 96-well plates in 100 µl RPMI-1640 supplemented with 10% FBS, and transfected with 50 nM miR-519 mimics or 50 nM negative control miR mimics (Genepharma Co., Ltd.) for 24, 48 and 72 h, as described above. MTT reagent (20 µl; Beijing Solarbio Science & Technology Co., Ltd.) was added to the wells 24 h post-transfection, prior to a 4-h incubation in darkness. The medium was then carefully discarded and blue formazan was dissolved with 200 µl dimethyl sulfoxide (Beijing Solarbio Science & Technology Co., Ltd.), and absorbance was measured at 550 nm (xMark, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Wells containing only HL60 cells served as controls.

Hoechst 33258 staining

The HL60 cells were cultured in 6-well plates at a density of 1×105 cells/well with DMEM (10% FBS). After 48 h transfection with miR-519 mimics, miR-519 inhibitor or negative control, the cells were washed with PBS and stained with 10 µg/ml Hoechst 33258 (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 5 min prior to being washed three times with PBS.

Immunofluorescence

The HL60 cells were cultured in 6-well chamber slides and fixed with 4% paraformaldehyde at a density of 1×105 cells/well for 10 min at −20°C. The slides were washed three times in PBS and incubated with a polyclonal antibody targeting HuR (cat. no. 12582) diluted in PBS (1:50) supplemented with 1% bovine serum albumin (BSA; Sigma-Aldrich) at 50 µl/slide for 2 h at room temperature. Following three washes with PBS (5 min each), the slides were incubated with tetramethylrhodamine-conjugated anti-rabbit IgG (1:100; cat. no. AP132R; Chemicon, Temecula, CA, USA). diluted in PBS (1:100) supplemented with 1% BSA at 50 µl/slide, for 1 h at room temperature. Following three washes with PBS, the slides were incubated with 10 µg/ml Hoechst 33258 for 5 min. The slides were washed again and examined using a fluorescence microscope (Leica CM3000; Leica Microsystems GmbH, Wetzlar, Germany).

Quantification of apoptotic cells

To quantify the number of apoptotic cells, flow cytometry was performed using an Annexin V-Fluorescein-5-Isothiocyanate (FITC) Apoptosis Detection kit (BioVision, Inc. Milpitas, CA, USA). A total of 48 h after transfection with 50 nM miR-519 mimics and 50 nM negative control, the HL60 cells were harvested in a 5-ml tube (Beijing Solarbio Science & Technology Co., Ltd.). The cells were then washed with cold PBS and resuspended at a final concentration of 1×106 cells/ml. FITC-Annexin V (5 µl) and propidium iodide (PI; 5 µl) were gently added and incubated with the cells for 15 min at a room temperature. Following incubation, the samples were analyzed by flow cytometry (EPICS® ALTRA™; BD Biosciences, Franklin Lakes, NJ, USA) within 1 h.

Inhibition of HuR by small interfering (si)RNA

HuR-specific siRNA (siHuR; 5′-aaggacgtagaagacatgt-3′) and negative control (5′-aagctctaattctgcaactct-3′) were purchased from Genepharma Co., Ltd. The cells were seeded at 1×105 cells/well in 6-well plates, and transfected with 50 nM siHuR or negative control for 48 h using a HiperFect transfection reagent (Qiagen China Co., Ltd.) as described above.

Cell migration assay

The cells were grown to confluence mono-layer in 6-well plates. To initiate migration, the cell layer was scratched using a pipette tip. The cells were subsequently transfected with antago-miR-519 (5′-CCAGAGGGAAGCGCCG-3′) or negative control (5′-CCATCAGTCCCAAATCCA-3′). Cell migration was captured under a microscope (CX21BIM-SET5; Olympus, Tokyo, Japan).

Statistical analysis

The data are presented as the mean ± standard error of the mean from three independent experiments. Statistical analysis was conducted with Student's t-test using Graphpad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

miR-519 is downregulated in ALM cells and tissue samples

RT-qPCR was used to quantify the expression levels of miR-519 in the HL60 human AML cells. U6 snRNA served as an internal control. miR-519 expression was significantly downregulated in >60% of HL60 cells, as compared with the NOMO1 cells (Fig. 1A; P<.0.05). The expression levels of miR-519 were then investigated in the tissue samples of 60 patients with AML, and 60 healthy control patients. The mean miR-519 expression levels were 0.39±0.17 and 0.96±0.21 for the AML and non-tumorigenic tissue samples, respectively. Therefore, miR-519 was downregulated in AML tissues compared with non-tumor tissues (Fig. 1B; P<.01).

Figure 1 Expression levels of miR-519 in AML cells and tissue samples. (A) RT-qPCR quantification of miR-519 expression in HL60 human AML and NOMO1 cells. (B) RT-qPCR quantification of miR-519 expression in the bone tissue samples of 60 patients with AML and 60 healthy (non tumor) patients. U6 served as an endogenous control. The data are presented as the mean ± standard error of the mean (n=6). *P<0.05 vs. the control group. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; AML, acute myeloid leukemia.

MiR-519 regulates HL60 cell viability

In order to determine the effects of miR-519 on cell viability, the HL60 cells were transfected with miR-519 mimics, miR-519 inhibitors or negative control for 24, 48, 72 h. As shown in Fig. 2A, upregulation of miR-519 expression decreased cell viability by 25 and 30% at 48 and 72 h, respectively, whereas downregulation of miR-519 expression increased cell viability in the HL60 cells by 20 and 30% at 48 and 72 h, respectively (Fig. 2B). These results suggest that miR-519 modulates HL60 cell viability.

Figure 2 miR-519 regulates HL60 human AML cell viability. The HL60 cells were transfected with miR-519 mimics, miR-519 inhibitors or NCs for 24, 48 and 72 h. (A) Upregulation of miR-519 expression decreased cell viability by 25 and 30%, at 48 and 72 h, respectively, whereas (B) downregulation of miR-519 expression increased cell viability in the HL60 cells by 20 and 30% at 48 and 72 h, respectively, as determined by MTT assay. The data are presented as the mean ± standard error of the mean (n=6) from independent experiments. *P<0.05 vs. NC. miR, microRNA; AML, acute myeloid leukemia; NC, negative control.

Upregulation of miR-519 induced HL60 cell apoptosis

The present study investigated the effects of miR-519 on cell apoptosis. Upregulation of miR-519 expression increased cell apoptosis by ~4-fold in the HL60 cells, as compared with the negative control (Fig. 3A). Cell morphology was also examined by Hoechst 33342 staining. As shown in Fig. 3B, the number of apoptotic cells increased in the HL60 cells transfected with miR-519 mimics, as compared with the negative control. These data suggest that upregulation of miR-519 induces HL60 cell apoptosis. Furthermore, western blot analysis was used to identify the molecular signaling pathway of cell apoptosis induced by miR-519 mimics. Upregulation of miR-519 expression significantly decreased the expression levels of HuR, and significantly increased the expression levels of Bax and Bcl-2-like protein 11 (Bim; Fig. 3C). miR-519 inhibitor was used to suppress miR-519 expression. As shown in Fig. 3D, suppression of miR-519 expression significantly increased the expression levels of HuR, and significantly decreased the expression levels of Bax and Bim. These results suggest that miR-519 may regulate HL60 cell apoptosis via modulating HuR, Bax and Bim expression.

Figure 3 Upregulation of miR-519 expression induces HL60 human AML cell apoptosis. miR-519 mimics or miR-519 inhibitors were transfected into the HL60 cells for 48 h. (A) Upregulation of miR-519 expression significantly increased the levels of cell apoptosis by 130% in the HL60 cells, as compared with the NC. (B) The number of apoptotic cells increased in the HL60 cells transfected with miR-519 mimics, as determined by Hoechst 33342 staining. The white arrows indicate the apoptotic cells (magnification, ×40). (C) As determined by western blotting, upregulation of miR-519 decreased the expression levels of HuR, and increased the expression levels of Bax and Bim, (D) whereas suppression of miR-519 expression elevated the expression levels of Bcl-2, and reduced the levels of Bax and Bim. The data are presented as the mean ± standard error of the mean (n=3) of independent experiments. *P<0.05 and **P<0.01 vs. NC. AML, acute myeloid leukemia; miR, microRNA; NC, negative control; HuR, human antigen R; Bcl-2, B cell lymphoma 2; Bax, Bcl-2-like protein 4; Bim, Bcl-2-like protein 11.

Overexpression of miR-519 inhibits cell growth, enhances apoptosis and decreases invasion of AML cancer cells

To investigate the role of miR-519 in AML, a cell migration assay was conducted by scratching the cell layer prior to transfection with antoga-miR-519 or negative control. Downregulation of miR-519 expression markedly enhanced cell migration (Fig. 4A). In addition, HuR-specific siRNA and Annexin V/PI were used to investigate the levels of cell apoptosis. Increased levels of cell apoptosis were detected in the HL60 cells transfected with HuR-specific siRNA, as compared with the control. Cell migration was also significantly reduced following HuR suppression (P<0.01; Fig. 4C).

Figure 4 miR-519 inhibited HL60 human AML cell migration predominantly by targeting HuR. (A) A cell migration assay was performed by scratching the cell layer prior to transfection with antago-miR-519 (As-miR-519) or NC. Time-based images were captured from 0–48 h. (B) Flow cytometric analysis of the HL60 cells transfected with HuR-specific siRNA. (C) Cell migration levels were analyzed following HuR inhibition. The data are presented as the mean ± standard error of the mean (n=3) from independent experiments. *P<0.05 and **P<0.01 vs. NC. AML, acute myeloid leukemia; si, small interfering; NC, negative control; miR, microRNA; HuR, human antigen R.

Discussion

The function and tissue specificity of miRs are under investigation in numerous diseases to further elucidate the underlying mechanisms of tumorigenesis. Previous studies suggested that miRs participate in numerous cell signaling pathways by targeting specific genes (25,26). In addition to miRs, ARE-mediated transcript degradation is an important post-transcriptional gene regulatory mechanism (12). AREs are cis-elements that control cell growth and proliferation, and numerous cancer-associated transcripts contain AREs in the 5′ or 3′-UTR, such as cytokines, growth factors and invasion factors (27). Via recognition of the AREs, RNA binding proteins regulate gene expression by altering the stability and translation efficiency of mRNA. The human ELAV protein, HuR, is an RNA-binding protein in the Hu family. HuR is widely expressed in mammalian cells and binds to AREs in order to regulate the stability of mRNAs; HuR is predominantly located in the nucleus of resting cells (28). A previous study reported that HuR binds to ARE-containing mRNAs and is transported into the cytoplasm. The translocation of HuR from the nucleus to the cytoplasm is an important mechanism underlying target mRNA stabilization (29).

A previous study demonstrated that miR-519 reduced cell proliferation by directly regulating HuR expression (21). The present study focussed primarily on miR-519, and investigated its ability to target HuR in AML. miR-519 is expressed at lower levels in AML cell lines, compared with healthy bone marrow cell lines. Overexpression of miR-519 was previously reported to decrease cell growth in murine ovarian tumors, and to control cell proliferation and clonogenic potential in ovarian surface epithelial cells (26), suggesting that miR-519 expression is significant in the underlying mechanisms of tumorigenesis in various cell types, potentially via regulation of HuR expression.

The present study establishes HuR as a target of miR-519 in AML. Furthermore, the restoration of miR-519 expression altered the leukemia phenotype, indicating the possible role of miR-519 as a tumor suppressor. To date, to the best of our knowledge, no genomic evidence has demonstrated miR-519 downregulation, other than observation of a decrease in functional HuR, which regulates miR-519 transcription in numerous types of cancer cell. These data lead to the hypothesis that myeloid leukemia cells may downregulate miR-519 to sustain HuR protein overexpression, resulting in leukemia progression.

The molecular mechanism underlying tumor suppression by miR-519 has been investigated in numerous types of cancer (21,26). Gene expression analyses have yet to be performed, suggesting that the cause of tumor suppression may be the ability of miRs to target genes associated with the cell cycle signaling pathway. To the best of our knowledge, HuR was the first direct miR-519 target gene to be identified (21). As a transcription factor, HuR regulates numerous genes that are known to contribute to normal cell life. The observed decrease in the expression levels of HuR target genes, such as Bax and Bim, in the present study may elucidate the cell cycle abnormalities exhibited by myeloid cell lines.

The results of the present study demonstrated that miR-519 targets HuR, mediating biological activity in normal and leukemic tissue samples. The reduced expression levels of miR-519 that deregulate HuR expression in hematopoietic development result in pathologic outcomes (primarily via upregulation of HuR protein expression), which leads to significant cell proliferation and survival. Therefore, restoration of miR-34b expression is potentially a fundamental step in treating AML and may provide a novel therapeutic strategy.

References