A total of 57 children with AML (28 male, 29 female; 10 months-14 years old) were identified after screening potential participants at the East Hospital of Shouguang People's Hospital (Shouguang, China) between January 2017 and January 2018. The inclusion criteria included a first-time diagnosis and no history of antitumour treatment. The exclusion criteria included the presence of other malignant tumours and/or organ dysfunctions. AML was diagnosed according to the World Health Organization classification of tumours of haematopoietic and lymphoid tissues (Version 2008) (24). A total of 57 age- and sex-matched children with normal bone marrow morphology, and without malignancy who were examined at the hospital at the same time were enrolled as the control group. The clinical characteristics of patients with AML and controls listed in Table I. The bone marrow samples were obtained by puncture and were stored at −80°C until use. Informed consent was obtained from the legal guardians of children <18 years old. This study was approved by the Ethics Committee of the East Hospital of Shouguang People's Hospital in accordance with the Declaration of Helsinki.

Human normal bone marrow CD34⁺ cells and AML cell lines (MV-4-11, AML-193, HL-60, and KG-1 cells) were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C. The small interfering RNA targeting SNHG14 (si-SNHG14) (5′-CAGCAUAUGUAAGUGGAACUCAGAA-3′), corresponding negative control (si-NC) (5′-AUCUUCAUUGGCACCGAACGUGUCACGUUU-3′), miR-193b-3p mimics (5′-AACUGGCCCUCAAAGUCCCGCU-3′), miR-193b-3p inhibitor (5′-AGCGGGACUUUGAGGGCCAGUU-3′), miR-NC (5′-UUUGUACUACACAAAAGUACUG-3′), pcDNA-SNHG14, pcDNA-NC, and pcDNA-MCL1 were all purchased from Shanghai GenePharma Co., Ltd. MV-4–11 and AML-193 cells were transfected with the aforementioned oligonucleotides (50 nM) or plasmids (50 ng) using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following an incubation period of 48 h at 37°C, the cells were utilised in the subsequent experiments.

Total RNA was isolated from the bone marrow samples and cells using an miRNeasy Mini kit (Qiagen GmbH). A PrimeScript™ IV 1st strand cDNA Synthesis Mix (Takara Bio, Inc.) was used to reverse transcribe the isolated RNA (2 µg) into cDNA according to the manufacturer's protocol. qPCR analyses were conducted using an SYBR Green PCR Master mix (Qiagen GmbH) on an ABI 7500HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc). The reaction conditions were as follows: 95°C for 3 min, and 40 cycles at 95°C for 10 sec, 60°C for 20 sec and 72°C for 34 sec. The melting curve had a single peak. The 2^−ΔΔCq method was used to analyse the relative mRNA expression levels (25). GAPDH or U6 was utilised as an internal control. The primer sequences were shown as follows: SNHG14 forward, 5′-GGGTGTTTACGTAGACCAGAACC-3′ and reverse, 5′-CTTCCAAAAGCCTTCTGCCTTAG-3′; miR-193b-3p forward, 5′-TCTACAGTGCACGTGTCTCCAG-3′ and reverse, 5′-ACCTGCGTAGGTAGTTTCATGT-3′; MCL1 forward, 5′-GGACATCAAAAACGAAGACG-3′ and reverse, 5′-GCAGCTTTCTTGGTTTATGG-3′; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-AGGGATCTCGCTCCTGGAA-3′; U6 forward, 5′-AGTACCAGTCTGTTGCTGG-3′ and reverse, 5′-TAATAGACCCGGATGTCTGGT-3′.

The targets of SNHG14 and miR-193b-3p were predicted by a starBase online database (starbase.sysu.edu.cn/agoClipRNA.php?). miR-193-3p was identified as a target of SNHG14, and MCL1 was identified as a target of miR-193b-3p.

The target association between SNHG14 and miR-193b-3p was analysed using the RIP assay with a Magna RIP RNA-binding protein immunoprecipitation kit (#17-700, EMD Millipore) in accordance with the manufacturer's instructions. Briefly, MV-4-11 and AML-193 cells were lysed in NP-40 lysis buffer containing 1% protease inhibitor (Sigma-Aldrich; Merck KGaA). After 10 min of centrifugation at 200 g and 4°C, 100 µl supernatant was conjugated with human anti-protein argonaute-2 (ab186733, 1:1,000, AGO2) antibody (Abcam) for 10 h at 4°C. Mouse IgG (M8642, 1:1,000, EMD Millipore) was used as a negative control. After Coprecipitated RNA was isolated and measured using RT-qPCR for direct binding as aforementioned.

The wild-type (wt) or mutated (mut) fragments of SNHG14 (SNHG14 wt/mut) and MCL1 (MCL1 wt/mut) were cloned into a pmiRGLO vector (Promega Corporation). To investigate the association among SNHG14, miR-193b-3p, and MCL1, MCL1 wt/mut or SNHG14 wt/mut was co-transfected into MV-4-11 and AML-193 cells with miR-NC/miR-193b-3p mimics using Lipofectamine^® 3000 reagent. After transfection for 48 h at 37°C, the Dual-Luciferase^® Reporter assay system (Promega Corporation) was used to measure the luciferase activity. Renilla luciferase activity was measured as the internal control.

MV-4-11 and AML-193 cells (1×10⁴ cells/well) were seeded in 96-well plates and cultured for 24, 48, 72 or 96 h. Cells were then incubated with 20 µl MTT (0.5 mg/ml) for 4 h at 37°C, followed by the addition of 150 µl dimethyl sulfoxide to terminate the reaction. The absorbance at 450 nm (A450) was measured using a microplate reader.

The cell apoptosis assay (MV-4-11 and AML-193 cells) was performed using an Annexin V-FITC/PI Apoptosis Detection kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, the cells were double-stained with 5 µl Annexin V/FITC and 10 µl PI. The cells were then incubated for 15 min at 25°C in the dark. The stained cells were detected using a flow cytometer (FACSAria II, Becton Dickinson, Franklin Lakes, NJ, USA) with CellQuest software (v1.5, Becton Dickinson).

Total proteins were isolated from MV-4-11 cells using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and quantified using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.). The proteins (30–50 µg/lane) were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene membrane. Following blocking with 5% non-fat milk for 2 h at 25°C, the membrane was incubated with rabbit anti-MCL1 (1:5,000; cat. no. ab246684; Abcam) and rabbit anti-β-actin (1:10,000; cat. no. 4970; Cell Signaling Technology, Inc.) primary antibodies at 4°C overnight. Then, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (A0545, 1:10,000; Sigma-Aldrich) for 1 h at room temperature. Finally, the protein bands were visualised using enhanced chemiluminescent substrate reagent kit (WP20005, Thermo Fisher Scientific, Inc.), and quantified using ImageJ software (v1.5, National Institutes of Health). β-actin was used as an internal control.

Each experiment was repeated at least three times. SPSS 22.0 statistical software (SPSS, Inc.) and GraphPad Prism v7.01 (GraphPad Software, Inc.) were used for all statistical analyses. Data are presented as the mean ± standard deviation. Student's t-test was used to compare significant differences between two groups and one-way ANOVA followed by Tukey's post hoc test was applied when analysing >two groups. Spearman's correlation analysis was performed to evaluate the correlation between SNHG14 and miR-193b-3p expression. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of SNHG14, the gene expression of SNHG14 was measured first. SNHG14 was found to be significantly overexpressed in AML bone marrow tissue compared with the level in normal marrow tissue (P<0.001; Fig. 1A). The AML bone marrow tissues were divided into two groups according to the expression of SNHG14: High expression, SNHG14 expression level ≥median and low expression, SNHG14 expression level <median. The clinicopathological features of the patients with AML are shown in Table II. The analysis of the data revealed that the French-American-British classification (P=0.348) and cytogenetics (P<0.001) were significantly different between the high and low expression groups (Table II). In addition, SNHG14 gene expression was significantly upregulated in AML cell lines (MV-4-11, AML-193, HL-60 and KG-1) compared with that in human normal bone marrow CD34⁺ cells (P<0.01; Fig. 1B). MV-4-11 and AML-193 cells, which had relatively higher SNHG14 gene expression, were utilised in the subsequent experiments.

To explore the effect of SNHG14 on AML progression, SNHG14 gene expression was knocked down by transfection of MV-4-11 and AML-193 cells with SNHG14 siRNA. RT-qPCR analysis revealed that SNHG14 expression was significantly reduced in the si-SNHG14-1 and si-SNHG14-2 groups compared with that in the blank control group (P<0.01; Fig. 2A). si-SNHG14-1 was utilised in the subsequent tests as it resulted in markedly lower SNHG14 expression compared with si-SNHG14-2. Cell viability was significantly lower in the si-SNHG14-1 group compared with that in the si-NC group for both cell lines (both P<0.05; Fig. 2B). Conversely, the apoptosis rate was significantly higher in the si-SNHG14-1 group compared with the si-NC group (P<0.01; Fig. 2C).

The starBase online database was used to predict the potential binding site between miR-193-3p and SNHG14 (Fig. 3A). As illustrated in Fig. 3B, SNHG14 knockdown significantly elevated miR-193-3p expression in MV-4-11 and AML-193 cells compared with the si-NC and blank control groups (all P<0.01). The results of the RIP assay indicated that the expression of SNHG14 and miR-193-3p were significantly enriched in the AGO2 immunoprecipitate compared with that in the IgG immunoprecipitate in both cell lines (all P<0.01; Fig. 3C). The DLR assay revealed that miR-193-3p mimics significantly decreased the luciferase activity of SNHG14 wt but did not affect that of SNHG14 mut in MV-4-11 and AML-193 cells (P<0.01; Fig. 3D). As shown in Fig. 3E, miR-193-3p expression was markedly decreased in bone marrow tissues from patients with AML compared to that in the NBM group (P<0.01). Additionally, a negative correlation was observed between the expression of miR-193-3p and SNHG14 (r=−0.4085; P=0.0016; Fig. 3F). miR-193-3p expression was significantly decreased in AML cell lines compared with that in human NBM CD34⁺ cells (P<0.01; Fig. 3G). Together, these results indicate that SNHG14 functions as a sponge for miR-193b-3p in AML cells.

As indicated in Fig. 4A, miR-193b-3p expression was markedly enhanced in the miR-193b-3p mimics group but was markedly decreased in the miR-193b-3p inhibitor group compared with that in the blank control group in both cell lines (both P<0.01). The MTT analysis revealed that miR-193b-3p mimics markedly reduced the viability and promoted the apoptosis of MV-4-11 and AML-193 cells compared with that in the miR-NC group (both P<0.01; Fig. 4B and C).

The starBase online database was used to first predict the possible binding site between miR-193b-3p and MCL1 (Fig. 5A). As shown in Fig. 5B, the DLR assay revealed that miR-193b-3p mimics significantly reduced the luciferase activity of MCL1 wt (P<0.05), whereas there was no notable change in the MCL1 mut in both cell lines. The expression of MCL1 was markedly elevated in bone marrow tissues from patients with AML compared with that in the NBM group (P<0.001; Fig. 5C). As shown in Fig. 5D and E, miR-193b-3p expression was negatively correlated with MCL1 (r=−0.4012; P=0.002), whereas SNHG14 expression was positively correlated with MCL1 (r=0.4322; P<0.001). Finally, the relative expression of MCL1 was significantly higher in all AML cell lines compared with that in CD34⁺ cells (P<0.01; Fig. 5F). Together, these results indicate that miR-193b-3p targets MCL1 and that MCL1 is highly expressed in AML.

The western blotting results indicated that the protein expression of MCL1 was notably decreased in the miR-193b-3p mimics + pcDNA-NC group compared with that in the miR-NC + PCDNA-NC group, and the decrease in MCL1 expression caused by miR-193b-3p was partially rescued by the overexpression of SNHG14 (P<0.05; Fig. 6A). To further investigate the underlying mechanism involved in the interaction among SNHG14, miR-193b-3p, and MCL1 in AML progression, MV-4-11 cells were co-transfected with si-SNHG14-1 and pcDNA-MCL1 or miR-193b-3p inhibitors. As shown in Fig 6B, MV-4-11 cell viability was significantly decreased in the si-SNHG14-1 group, and the decrease in cell viability induced by SNHG14 knockdown was recovered by transfection with pcDNA-MCL1 or miR-193b-3p inhibitors (P<0.05). Conversely, MV-4-11 cell apoptosis was markedly enhanced in the si-SNHG14-1 group, and pcDNA-MCL1 or miR-193b-3p inhibitors partially eliminated the increase in cell apoptosis caused by SNHG14 knockdown (P<0.05; Fig. 6C). In summary, these results indicate that SNHG14 may act as ceRNA to modulate AML cell viability and apoptosis via the miR-193b-3p/MCL axis.