A total of 40 bone marrow specimens were collected from patients with AML (mean age, 62.3 years; age range, 32 to 71 years) at the Affiliated Hangzhou First People's Hospital from May 2016 to Feb 2020. Bone marrow specimens were obtained from 40 healthy volunteers (mean age, 60.3 years; age range, 36 to 72 years) at the Affiliated Hangzhou First People's Hospital from May 2018 to Feb 2020. The present study was approved by the Ethics Committee of Affiliated Hangzhou First People's Hospital (Hangzhou, China; approval no. 146-01). Written informed consent was provided by all patients prior to the study start. The clinical characteristics of the patients are presented in Tables I and II. AML HL-60 and THP-1 cell lines and the normal BM HS cell line were supplied by the American Type Culture Collection. All cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured in a humidified incubator with 5% CO₂ at 37°C.

pcDNA3.1/LINC01018, a miR-499a-5p mimic (5′-UUAAGACUUGCAGUGAUGUUU-3′), miR-499a-5p inhibitor (5′-AAACATCTCTGCAAGTCTTAA-3′), miR mimic control (5′-ACUACUGAGUGACAGUAGA-3′), miR inhibitor control (5′-CAGUACUUUUGUGUAGUACAA-3′), si-PDCD4 (sense, 5′-GGAGCGGUUUGUAGAAGAAdTdT-3′ and antisense, 3′-dTdTCCUCGCCAAACAUCUUCUU-5′), si-control (sense, 5′-UUCCCUUUGUCAUCCUUUGCCUdTdT-3′ and antisense, 3′-dTdTAAGGGAAACAGUAGGAAACGGA-5′) and a pcDNA3.1 empty vector were all purchased from Shanghai GenePharma Co., Ltd. HL-60 and THP-1 cells were transfected with pcDNA3.1 (50 ng), pcDNA3.1/LINC01018 (50 ng), miR-499a-5p mimic (100 nM), mimic control (100 nM), miR-499a-5p inhibitor (100 nM), inhibitor control (100 nM), si-PDCD4 (50 nM), and si-control (50 nM) at 37°C for 48 h using Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected and used 48 h later for subsequent experimentation.

Total RNA from AML tissues and cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and its quality was detected via NanoDrop 2000c (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Next, the RNA samples were converted into cDNA using M-MLV reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The reverse transcription protocol was 70°C for 10 min followed by 2 min on ice. RT-qPCR was conducted on an ABI 7900 system with SYBR Green Real-Time PCR master mixes (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. GAPDH was used as an internal control for LINC01018 and PDCD4, and U6 small nuclear RNA served as a control for miR-499a-5p. The relative expression was analyzed using the 2^−ΔΔCq method (33). The sequences of all primers are presented in Table III.

The effects of LINC01018 and miR-499a-5p on AML cell viability were evaluated using the Cell Counting kit-8 (CCK-8; Sigma-Aldrich; Merck KGaA) assay. AML cells were seeded onto 96-well plates (4×10³) and cultured overnight at 37°C followed by transfection of the indicated oligonucleotides. After 12, 24, 48 and 72 h of transfection, the absorbance of each well was detected at 450 nm by a spectrophotometer. The results represent the mean of three replicates under the same conditions.

AML cell proliferation was analyzed by an EdU assay kit (Guangzhou RiboBio Co., Ltd.), according to the manufacturer's protocols. Cell nuclei were stained with Hoechst 3344 (4 µg/ml, Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 30 min. EdU-positive cells were red.

Apoptosis of AML cells was detected by TUNEL staining using a cell death detection reagent, fluorescein (Roche Diagnostics). Briefly, transfected AML cells (1×10⁶) were added into the slides with polylysine. Cells were fixed with 2% formaldehyde at 4°C for 30 min and permeabilized using 0.2% Triton-X. After washing three times with PBS, cells were processed with 20 µg/ml Proteinase K (Sigma-Aldrich; Merck KGaA; cat. no. P2308) at room temperature for 20 min. Next, cells were washed with PBS three times followed by a 1 h incubation of the TUNEL reaction mixture at 37°C according to the manufacturer's instructions. Nucleic acids were stained using DAPI (1:500, Sigma-Aldrich, cat. no. D9564) for 15 min at room temperature. Subsequently, cells were incubated with 50 µl DAB solution at room temperature for 20 min, and the nuclei were stained using hematoxylin at room temperature for 3 min. After dehydration and transparency, the signals were scanned from 6 random fields using a confocal microscope (Olympus BX51TRF; Olympus Corporation) (magnification, ×100) and the number of TUNEL-positive cells was counted. The apoptosis index was used to measure the degree of cell apoptosis.

RIP was performed to analyze the interplay between LINC01018 and miR-499a-5p using an EZ-Magna RIP kit (Merck KGaA). Following cell lysis, the cell extract was incubated with RIP buffer containing an AGO2 antibody coated on magnetic beads, and an IgG antibody was used as a control. The precipitated RNAs were quantified and purified and then reverse transcribed into cDNA, and RT-qPCR was performed to analyze LINC01018 and miR-499a-5p levels.

Total protein was extracted from HL-60 and THP-1 cells using cell lysis buffer (cat. no. AR0105; Wuhan Boster Biological Technology, Ltd.). Following the protein concentration being determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), 50 µg samples were isolated by 10% SDS-PAGE gels, transferred onto PVDF membranes (Merck KGaA) and blocked with 5% skimmed milk for 1 h at room temperature. Subsequently, the membranes were incubated with Bax antibody (dilution, 1:1,000; cat. no., ab182733; Abcam) or Bcl-2 antibody (dilution, 1:1,000; cat. no., ab32124; Abcam) at 4°C overnight, followed by incubation with the corresponding HRP-conjugated secondary antibodies (1:2,000; cat. nos. ab205719 or ab6721; Abcam) at room temperature for 1 h. Finally, the protein bands were visualized using an enhanced chemiluminescence reagent (Merck KGaA) and GAPDH was used as an internal control.

miR-499a-5p information was obtained through miR Base (http://www.mirbase.org/index.shtml). The prediction of LINC01018 and miR-499a-5p was conducted using starbase v2.0 (http://starbase.sysu.edu.cn). The target genes including PDCD4 of miR-499a-5p were predicted using starbase v2.0, TargetScan (http://www.targetscan.org/) and MicroRanda (http://www.microrna.org/).

The interaction between miR-499a-5p and LINC01018 or PDCD4 was verified by dual-luciferase assays. In brief, the wild-type (WT) and mutant (MUT) sequences of LINC01018 or PDCD4, which contained miR-499a-5p binding sites, were amplified and inserted into pmirGLO (Promega Corporation). The recombinant luciferase plasmids were named LINC01018-WT, PDCD4-WT1, PDCD4-WT2, PDCD4-WT3, LINC01018-MUT, PDCD4-MUT1, PDCD4-MUT2, PDCD4-MUT3. AML cells were co-transfected with miR-499a-5p mimics or negative control and the indicated recombinant luciferase plasmids (0.8 µg) and 0.08 µg renilla plasmid (RL-SV40; Promega Corporation) using Lipofectamine 3000^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of transfection, the luciferase activity was analyzed by a dual-luciferase reporter assay system (Promega Corporation) according to the manufacturer's instructions. The results were normalized using Renilla luciferase activity.

All results are presented as the mean ± standard deviation. The two groups were compared using the unpaired Student's t-test, and multiple comparisons were conducted using one-way analysis of variance, followed by Tukey's post hoc test in GraphPad Prism 7 (GraphPad Software, Inc.). Correlation analysis was conducted using the Pearson correlation coefficient. P<0.05 was considered to indicate a statistically significant difference.

RNAalifold Web Server was used to obtain the predicted secondary structures of LINC01018: Centroid and minimum free energy predictions. A mountain plot of the secondary structures is shown in Fig. 1A. The expression of LINC01018 and miR-499a-5p in AML tissues and cell lines was measured using RT-qPCR. Compared with that in healthy controls (n=40), the expression level of LINC01018 was decreased, while miR-499a-5p was increased, in AML tissues (n=40; P<0.05; Fig. 1B and C). The results in Fig. 1D show a negative correlation between LINC01018 and miR-499a-5p expression in patients with AML (r=−0.3881; P=0.0134). In addition, RT-qPCR analysis indicated that LINC01018 levels were markedly decreased in HL-60 and THP-1 cell lines compared with that in HS cells (P<0.05; Fig. 1E). The expression of miR-499a-5p was significantly higher in HL-60 and THP-1 cells than in HS cells (P<0.05, Fig. 1F). These data demonstrated that LINC01018 was expressed at low levels, while miR-499a-5p was highly expressed, in AML tissues and cell lines.

Following investigating the levels of LINC01018 and miR-499a-5p and verifying their correlation, the biological functions of miR-499a-5p and LINC01018 in AML were analyzed. To begin with, RT-qPCR was applied to investigate the overexpression efficiency of LINC01018 in AML cells. The results indicated that the LINC01018 level was significantly higher in the pcDNA3.1/LINC01018 group than in the blank and negative control groups (P<0.05; Fig. 2A). Next, CCK-8 and EdU staining assays were performed to detect the proliferation of HL-60 and THP-1 cells following transfection of LINC01018 alone or LINC01018 plus miR-499a-5p. The results revealed that LINC01018 transfection significantly suppressed cell proliferation; however, this effect was reversed by the overexpression of miR-499a-5p (P<0.05; Fig. 2B-F). Furthermore, the TUNEL assay verified that LINC01018-overexpression increased the apoptosis rate of HL-60 and THP-1 cells, while miR-499a-5p overexpression reversed this effect (P<0.05; Fig. 2G-I). In addition, western blot analysis demonstrated that the protein expression levels of Bax and Bcl-2 were increased in HL-60 and THP-1 cells following transfection with pcDNA3.1/LINC01018, while miR-499a-5p overexpression abolished this upregulation of Bax and Bcl-2 (P<0.05; Fig. 2J-K). In conclusion, LINC01018-overexpression inhibited the growth of HL-60 and THP-1 cells; however, miR-499a-5p blocked these effects.

To further study the mechanism of LINC01018 and miR-499a-5p in AML, the association between LINC01018 and miR-499a-5p and the potential targeted genes of miR-499a-5p was investigated by bioinformatics analysis. To begin with, the transfections were successful with miR-499a-5p or miR-499a-5p inhibitors in all cell types validated by qPCR (Fig. 3A and B). One complementary binding site was identified between LINC01018 and miR-499a-5p (Fig. 3C; upper panel). The luciferase reporter assay results demonstrated that the transfection of miR-499a-5p mimics strongly decreased the luciferase activity of cells following transfection with LINC01018-WT (P<0.05; Fig. 3C; lower panel). Furthermore, three binding sites between miR-499a-5p and PDCD4 were predicted using a bioinformatics tool (Fig. 3D; upper panel). The luciferase reporter results demonstrated that the luciferase activities were markedly decreased following co-transfection with miR-499a-5p and PDCD4-WT1, PDCD4-WT2 or PDCD4-WT3 relative to NC mimics, while co-transfection with miR-499a-5p and PDCD4-MUT1, PDCD4-MUT2 or PDCD4-MUT3 caused no notable change relative to NC mimics (P<0.05; Fig. 3D; lower panel). Next, an RNA pull-down assay was performed to investigate the interaction between LINC01018 and miR-499a-5p. Compared with that in the Bio-probe-NC group, miR-499a-5p was significantly enriched in the Bio-WT-LINC01018 group but not in the Bio-MUT-LINC01018 group (P<0.05; Fig. 3E). Furthermore, a RIP assay was used to further confirm the interplay between LINC01018 and miR-499a-5p. The results indicated that LINC01018 and miR-499a-5p were significantly enriched in the anti-AGO2 group compared with the anti-IgG group (P<0.05; Fig. 3F). In addition, western blotting was performed to measure PDCD4 protein expression. The expression of PDCD4 was markedly increased in HL-60 and THP-1 cells following knockdown of miR-499a-5p or overexpression of LINC01018 relative to the negative control (Fig. 3G). Taken together, these results indicated that PDCD4 is a target gene of miR-499a-5p and that its expression level is regulated by LINC01018 through miR-499a-5p.

Following confirming the result that PDCD4 was a target gene of miR-499a-5p, the effects of PDCD4 on miR-499a-5p were investigated. To begin with, a PDCD4-knockdown siRNA was designed and its efficiency was validated (Fig. 4A). Next, western blot analysis of PDCD4 protein expression demonstrated that PDCD4 was significantly increased by transfection of HL-60 and THP-1 cells with a miR-499a-5p inhibitor, while transfection of si-PDCD4 reversed this effect (P<0.05; Fig. 4B). The EdU proliferation assay was performed to determine the effects of co-transfection of miR-499a-5p inhibitor and si-PDCD4 on cell proliferation. The results demonstrated that knockdown of miR-499a-5p inhibited the proliferation of HL-60 and THP-1 cells compared with the blank and negative controls, while si-PDCD4 transfection abolished the inhibitory effects of miR-499a-5p on cell proliferation (P<0.05; Fig. 4C and D). TUNEL assay results demonstrated that the apoptosis rate was upregulated by knockdown of miR-499a-5p in HL-60 and THP-1 cells, while transfection of si-PDCD4 reversed the promotive effects of miR-499a-5p-knockdown on cell apoptosis (P<0.05; Fig. 4E and F). Furthermore, the expression levels of Bax and Bcl-2 were increased by miR-499a-5p inhibitor transfection, while co-transfection of miR-499a-5p inhibitor and si-PDCD4 abrogated this effect (P<0.05; Fig. 4G and H). These data demonstrated that the miR-499a-5p inhibitor suppressed the proliferation of HL-60 and THP-1 cells but promoted cell apoptosis, which was accomplished through regulation of PDCD4.

To investigate the correlation between PDCD4 and LINC01018 or miR-499a-5p, the expression level of PDCD4 was measured by RT-qPCR. The results demonstrated that PDCD4 expression was downregulated in HL-60 and THP-1 cells (P<0.05; Fig. 5A) and AML tissues (P<0.05; Fig. 5B), compared with HS cells and healthy controls. In addition, the results in Fig. 5C demonstrated a positive correlation between PDCD4 and LINC01018 expression levels in patients with AML (r=0.5098; P=0.0008). By contrast, PDCD4 and miR-499a-5p were negatively correlated in patients with AML (r=−0.3342; P=0.0296; Fig. 5D). In summary, these results suggested that LINC01018 contributed toward AML cell growth by modulating PDCD4 through suppression of miR-499a-5p (Fig. 5E).