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Introduction

Acute myeloid leukaemia (AML) is a heterogeneous haematopoietic malignancy, which is well-known for the rapid accumulation and malignant proliferation of immature myeloid progenitors in the bone marrow (BM) and peripheral blood (PB) (1). For decades, without efficient therapeutic strategy, AML quickly led to poor prognosis and became fatal. Over the last few decades, AML treatment has improved in risk assessment, post-remission chemotherapy and haematopoietic stem-cell transplantation (2,3). The phosphoinositide 3-kinase, AKT, and mammalian target of rapamycin (PI3K/AKT/mTOR) signalling network controls proliferation, differentiation, and survival of haematopoietic cells (4,5). The fact that in 50–80% of AML cases, the PI3K/AKT/mTOR pathway is abnormally activated indicates that the PI3K/AKT/mTOR pathway is a promising therapeutic target (6,7), and many agents have been developed. LY294002, which directly targets PI3K, has revealed strong cytotoxic effects in preclinical AML models (8–10). Ribavirin, an eIF4E inhibitor, was studied in preclinical models and in a pilot study and presents efficiency in 30% of AML patients (11,12). Aberrant activation of AKT is the most common frequent cause in AML, and dual PI3K/mTOR inhibitors (P-103 and BEZ235), mTOR1/2 inhibitors (OSI-027 and PP242), and AKT inhibitors (perifosine) were also investigated in preclinical models (13–17).

Long non-coding RNAs (lncRNAs) are a class of RNA molecules >200 nucleotides. They are transcribed and not protein coded. A large number of this class of RNA molecules has been identified, numbering >58,000 (18,19). Accumulating evidence has revealed that lncRNAs play critical roles not only in physiological processes in normal cells but also in a multitude of biological processes that are central in tumorigenesis and the progression of cancer (18,20), including in AML. Wu et al reported that HOX transcript antisense intergenic RNA (HOTAIR) was overexpressed in AML and was a promising biomarker of poor prognosis (21). By performing large-scale sequencing, it was determined via evaluation of the lncRNA profile that it is possible to provide valuable information for improved risk stratification of AML patients (22).

lncRNAs are reported to regulate the PI3K/AKT/mTOR pathway in several types of cancer cells. Highly upregulated in liver cancer (HULC) was revealed to act as an oncogene in gliomas (23). The upregulated HULC promoted malignant cell behaviours, including proliferation, migration and invasion via activation of the PI3K/AKT/mTOR pathway in glioma cells (23). A newly identified lncRNA ENST00113, which is most upregulated in atherosclerosis, regulated proliferation, survival and migration by activating the PI3K/AKT/mTOR pathway, potentially by inhibiting the process of phosphorylation (24). lncRNA LUNAR1 was reported to play a central role in the interplay of NOTCH1-IGF-1 interactions via activation of the PI3K/AKT/mTOR pathway in AML cells (25). Accordingly, these data revealed that lncRNAs are tightly associated with activation or inactivation of the PI3K/AKT/mTOR pathway and thus play a regulatory role in the malignant behaviours of several types of cancers, including AML.

Long intergenic non-protein coding RNA 239 (LINC00239) is a newly identified lncRNA that has been revealed to be aberrantly expressed in AML (26), without knowledge of its exact role in AML. In the present study, the fundamental role of LINC00239 in human AML cells was examined and the potential role in regulating malignant behaviours via the PI3K/AKT/mTOR pathway was investigated.

Materials and methods

Cell culture and treatment

The AML cell lines HL-60 (FAB M2) and KG-1 (erythroleukemia-FAB M6) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% antibiotic-antimycotic mixture (Thermo Fisher Scientific, Inc.) in a humidified, 37°C and 5% CO2 atmosphere. Medium was half-refreshed every 3 days.

Doxorubicin and dual pan-PI3K/mTOR kinase inhibitor dactolisib (NVP-BEZ235) were purchased from (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). For the evaluation of chemosensitivity to doxorubicin, a range concentration from 0.5 to 100 µM was incubated with KG-1 or HL-60 cells for 24 h, respectively. For inhibition of the PI3K/AKT/mTOR pathway, KG-1 or HL-60 cells were pretreated with 0.5 Μm NVP-BEZ235 for 6 h.

RT-qPCR

Total RNA was isolated from culture cell using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription of RNA was performed following the instructions of the MBI Fermentas kit (Fermentas; Thermo Fisher Scientific, Inc.). Reaction conditions were as follows: 70°C for 10 min, ice bath for 2 min, 42°C for 60 min and 70°C for 10 min. The cDNA was stored in a −80°C freezer. The reaction system for qPCR was prepared following the instructions of the kit (Fermentas; Thermo Fisher Scientific, Inc.). The primer sequences were as follows: linc00239, forward 5′-AGGTACCAGCTGGGATGTTG-3′ and reverse 5′-CATGACCGTCTTCCTGTGTG-3′; U6, forward 5′-GTGCTCGCTTCGGCAGCACA-3′ and reverse 5′-AAAATATGGAACGCTTCA-3′; β-actin, forward 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse 5′-CTCCTTAATGTCACGCACGAT-3′; GAPDH, forward 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. The reaction conditions were as follows: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 5 sec, annealing and extension at 60°C for 1 min, 35 cycles in total. snoU6, β-actin and GAPDH were used as the internal references. The expression of the genes were calculated using relative quantitative measurement, where 2−ΔΔCq represented the relative expression of each target gene (27). Each experiment was repeated for 3 times.

Cell viability assays

Cells were seeded (5×103/well) into 96-well plates overnight. After treatment with doxorubicin (0.5–100 µM) for 24 h, 10 µl tetrazolium salt Cell Counting Kit-8 (CCK-8) (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was added to each well (final volume ratio, 10%). Optical density (OD) was assessed at a wavelength of 450 nm (OD450).

Cell cycle distribution

Cells were pelleted and washed with pre-cold phosphate-buffered saline (PBS) 3 times. Cells were fixed with 1 ml of 75% ethyl alcohol, stored overnight at 4°C, washed with PBS 2 times and 100 µl RNase A and 400 µl propidium iodide (PI; Sigma-Aldrich; Merck KGaA) were added in the dark and incubated for 30 min at room temperature. After 30-min incubation at 4°C, flow cytometric analysis was performed using the FACS LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Colony formation on soft agar

KG-1 or HL-60 cells (5×103) were suspended in serum-free RPMI-1640 medium and dissolved in 0.3% low-melting agarose (Sigma-Aldrich; Merck KGaA), supplemented with 10% FBS and a 1% antibiotic-antimycotic mixture. Cells were allowed to grow for 14 days and colonies were identified as having >50 cells. Cells were visualized using an Olympus CX41 microscope (Olympus Corp., Tokyo, Japan).

Migration

Transwell permeable plates with 8.0 µM pores (Corning Incorporated Life Sciences, Corning, NY, USA) were employed to analyze cell migration. In the bottom chamber, 500 µl of serum-free RPMI-1640 medium was added supplemented with 5% FBS. In the upper chamber, 5×104 cells were added suspended in serum-free medium. After 24 h of incubation, the amount of viable migrated cells was determined by counting using trypan blue staining. Cells were visualized using an Olympus CX41 microscope (Olympus Corp.).

CFSE/PI staining

Cells were washed in serum-free RPMI-1640 medium 3 times and suspended in 500 µl of fresh medium. Carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescent dye (100 µl) (5 µmol/l) was added and incubated at 37°C for 30 min. Then, the cells were washed with fresh medium 3 times. Subsequently, 24 h later, cells were collected and suspended in 500 µl fresh medium and incubated with 5 µg/ml PI at room temperature for 10 min. Flow cytometry was performed using the FACS LSR II flow cytometer (BD Biosciences). Analysis of results was performed using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA). The experiments were repeated at least 3 times.

Annexin V-FITC/PI staining

KG-1 or HL-60 cells were washed with 1 ml of ice-cold PBS 3 times. Cells were suspended with 100 µl of binding buffer, 5 µl of Annexin V-FITC (BD Biosciences) and 10 µl of PI (Sigma-Aldrich; Merck KGaA) for 10 min avoiding light at room temperature. Then, FITC and PI signals were assessed using FACS LSR II flow cytometer (BD Biosciences). Analysis of results was performed using FlowJo 7.6 software (Tree Star, Inc.). The experiments were repeated at least 3 times.

TUNEL staining

Cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After 3 washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were stained with In Situ Cell Death Detection Kit (Roche Diagnostics, Basel, Switzerland) following the manufacturer's instructions. Cells were visualized under an X71 (U-RFL-T) fluorescence microscope (Olympus, Melville, NY, USA) and analyzed with ImageJ software (version 1.46; National Institutes of Health, Bethesda, MD, USA).

Western blotting

For the extraction of total protein from KG-1 or HL-60 cells, 100 µl of lysis buffer containing 1% NP-40, 100 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM EDTA was employed. The protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). Total protein (20 µg) was fractionated in a 10% SDS gel and transferred to nitrocellulose membranes. The membranes were blocked in PBS with 0.25% Tween-20 (PBS-T) containing 5% skim milk for 1 h at room temperature. The blocked membranes were incubated with primary antibodies for 2 h at room temperature. The primary antibodies used were as follows: anti-γ-H2A.X antibody diluted in 1:1,000 (cat. no. ab2893), anti-histone H2A.X antibody diluted in 1:2,500 (cat. no. ab11175), anti-β-actin antibody diluted in 1:5,000 (cat. no. ab8226), anti-AKT (phospho S473) antibody diluted in 1:1,000 (cat. no. ab18206), anti-pan-AKT antibody diluted in 1:1,000 (cat. no. ab8805), anti-mTOR (phospho S2448) diluted in 1:500 (cat. no. ab109268), anti-mTOR diluted in 1:1,000 (cat. no. ab2732) and anti-GAPDH antibody diluted in 1:5,000 (cat. no. ab8245). All antibodies were purchased from Abcam (Cambridge, UK). After 3 washes with PBS-T, the membranes were incubated with the corresponding following secondary antibodies: HRP-labeled goat anti-mouse IgG antibody diluted in 1:5,000 (cat. no. ab97040) or goat anti-rabbit IgG antibody diluted in 1:5,000 (cat. no. ab7090) for 2 h at room temperature. Protein levels were detected after incubation with SuperSignal Wester Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.). Blots were imaged and analyzed with Quantity One (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis

All data were presented as the means ± SD (SD). Student's t-test was performed to assess statistical significance. The Wilcoxon signed-rank test was used to compare paired samples, whereas Kruskal-Wallis and Mann-Whitney U tests were used to compare different groups followed by the Tukey's post hoc test. P-values <0.05 were considered to indicate a statistically significant difference.

Results

Confirmation of the existence of lncRNA linc00239 in the AML cell lines KG-1 and HL-60

In order to identify the expression levels of linc00239 in the AML cell lines KG-1 and HL-60, RT-qPCR was performed, and the data were normalized to β-actin, GAPDH or snoU6 RNA. As revealed in Fig. 1A, the expression of linc00239 was detected in both KG-1 and HL-60 cells, and KG-1 presented an evidently higher expression compared to HL-60. Northern blotting also confirmed the existence of linc00239 in both of these cell lines (Fig. 1B). shRNA targeted to linc00239 (shlinc00239) or scrambled shRNA (shScrambled) was transiently transfected into KG-1 cells for linc00239 knockdown. linc00239 expression vector (linc00239) or empty vector (vector) was transiently transfected into HL-60 cells for linc00239 overexpression analysis. Efficient overexpression by expressing vector or knockdown by shlinc00239 transient transfection was confirmed by RT-qPCR (Fig. 1C).

Figure 1. Detection of the expression levels of linc00239 in KG-1 and HL-60 cells (A) RT-qPCR was performed to detect the linc00239 levels in KG-1 and HL-60 cells, which were normalized to the expression levels of β-actin, GAPDH or snoU6. (B) Northern blotting was performed to detect the existence of linc00239 in KG-1 and HL-60 cells. (C) After introduction of siRNA or expression vector separately, the difference in linc00239 levels was detected by RT-qPCR. *P<0.05 vs. shScrambled-transfected KG-1 cells; #P<0.05 vs. vector-transfected HL-60 cells. linc00239, long intergenic non-protein coding RNA 239.

Effects of linc00239 on cell viability, cell cycle distribution, colony formation and migration

The accumulating evidence indicating that lncRNAs may tightly regulate tumour development encouraged us to investigate the exact role of linc00239 in AML cells (28,29). By performing a CCK-8 assay and PI staining, followed by a flow cytometric assay, the results revealed that although linc00239 knockdown slightly affected cell viability and cell cycle distribution in KG-1 cells, linc00239 overexpression significantly promoted cell viability potentially by promoting the cell cycle by decreasing the G1/G0 phase in HL-60 cells (Fig. 2A and B). The effects of linc00239 on colony formation and migration were further detected to illustrate the role of linc00239 in the regulation of malignant behaviours in AML cells. As revealed in Fig. 2C and D, in both KG-1 and HL-60 cells, the presence of linc00239 positively regulated colony formation and migration.

Figure 2. Effects of linc00239 on proliferation, colony formation, cell cycle distribution, migration and apoptosis. (A) A CCK-8 assay was performed to assess the effect of modified linc00239 in KG-1 or HL-60 cells, respectively. *P<0.05 vs. vector-transfected HL-60 cells. (B) The effects of knockdown or overexpression of linc00239 on cell cycle phases. *P<0.05 vs. vector-transfected HL-60 cells. (C) Colony formation in soft agar was performed after knockdown or overexpression of linc00239. (D) A migration assay was performed to detect the effects of expressed linc00239 on cell migration. *P<0.05 vs. shScrambled-transfected KG-1; #P<0.05 vs. vector-transfected HL-60 cells. linc00239, long intergenic non-protein coding RNA 239; CCK-8, Cell Counting Kit-8.

linc00239 regulates chemoresistance to doxorubicin and exerts a protective effect against apoptotic cell death

Various lncRNAs have been reported to regulate chemosensitivity in cancer cells to therapeutic agents, including doxorubicin, by modulating DNA damage response pathways (30,31). This encouraged us to investigate the role of linc00239 in the chemosensitivity to doxorubicin, which is a commonly used therapeutic agent. By exposure to a concentration range of doxorubicin, relative cell viability was assessed by performing a CCK-8 assay. In KG-1 cells, knockdown of linc00239 significantly decreased the IC30 and IC50 concentrations of doxorubicin (Fig. 3A and B, upper panel). After 1.75 µM treatment of doxorubicin, the rate of cell death detected by CFSE/PI double staining illustrated consistently that knockdown of linc00239 significantly increased the cell death rate. In HL-60 cells, overexpression of linc00239 significantly increased the IC30 and IC50 concentrations of doxorubicin (Fig. 3A and B, lower panel). As anticipated, overexpressed linc00239 decreased the rate of cell death after 2.68 µM treatment of doxorubicin (Fig. 2B).

Figure 3. Effects of linc00239 on the chemosensitivity of KG-1 or HL-60 to doxorubicin. Chemosensitivity to doxorubicin was measured by the (A) cell viability assay, and (B) IC30 and IC50 were assessed separately. *P<0.05 vs. shScrambled-transfected KG-1 cells; #P<0.05 vs. vector-transfected HL-60 cells. linc00239, long intergenic non-protein coding RNA 239.

Doxorubicin exerts an anticancer effect mainly via induction of apoptotic cell death (32). To further confirm the effects of linc00239 on doxorubicin-induced apoptotic cell death and DNA damage, apoptotic cell death and the level of phosphorylation (Ser139) and total histone H2AX protein by Annexin V-FITC/PI double staining were assessed, followed by flow cytometric assay and western blotting. These data revealed that in KG-1, knockdown of linc00239 significantly increased the proportion of Annexin V-FITC+/PI− and Annexin V-FITC+/PI+ (Fig. 4A). Since KG-1 cells presented a much higher expression level of linc00239 compared to HL-60 cells, linc00239 was knocked down in KG-1 and overexpressed linc00239 in HL-60 cells. In HL-60 cells, overexpression of linc00239 significantly decreased the proportion of Annexin V-FITC+/PI−. Detection of phosphorylation (Ser139) and total histone H2AX protein illustrated that the overexpression of linc00239 inhibited phosphorylation (Ser139) of histone H2AX without disturbing total histone H2AX (Fig. 4B). To elucidate whether linc00239 had a protective effect against DNA damage in KG-1 and HL-60 cells, a terminal dUTP nick-end labelling (TUNEL) assay was performed. The results clearly revealed a decrease in the percentage of TUNEL-positive cells, confirming the protective effect against doxorubicin-induced DNA damage (Fig. 4C).

Figure 4. Downregulated or upregulated linc00239 affects doxorubicin-induced apoptotic cell death. (A) Annexin V-FITC/PI double staining was performed, followed by flow cytometric analysis. *P<0.05 vs. shScrambled-transfected KG-1 cells; #P<0.05 vs. vector-transfected HL-60 cells. (B) Western blotting was performed to detect H2AX and phosphorylated H2AX after doxorubicin treatment. (C) The TUNEL assay was performed to visualize apoptotic cells after doxorubicin treatment. linc00239, long intergenic non-protein coding RNA 239; PI, propidium iodide.

The presence of linc00239 is tightly associated with activation of the PI3K/Akt/mTOR pathway

To assess whether the expression of linc00239 was associated with activation of the PI3K/Akt/mTOR pathway, the phosphorylation of Akt and mTOR was detected after linc00239 knockdown in KG-1 cells or overexpression in HL-60 cells. Forty-eight hours after transfection, protein lysates were obtained and analysed by western blotting. The results revealed that knockdown of linc00239 reduced the phospho/total Akt ratio in KG-1 cells, and in contrast, overexpression of linc00239 significantly increased the phospho/total Akt ratio in L-60 (Fig. 5A). Moreover, the expression level of linc00239 also positively regulated the phosphorylation levels of mTOR on S2448, thus affecting mTORC1 activity, without disturbing mTOR total protein. To further confirm whether linc00239 regulated the PI3K/Akt/mTOR pathway by promoting phosphorylation of Akt and mTOR, the dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 was employed. After pretreatment with 0.5 µM NVP-BEZ235, phosphorylation of Akt and mTOR were inhibited, and the phosphorylation-promoting effect of linc00239 was abolished (Fig. 5B).

Figure 5. linc00239 contributes to PI3K/Akt/mTOR pathway activation in both KG-1 and HL-60 cells. (A) After introduction of shlinc00239 or linc00239 expression vectors, the total amount of Akt, mTOR, or phosphorylated Akt and mTOR was assessed. *P<0.05 vs. shScrambled-transfected KG-1 cells; #P<0.05 vs. vector-transfected HL-60 cells. (B) After 1 µM BEZ treatment, the activating effects of ectopic linc00239 on the PI3K/Akt/mTOR pathway were assessed by western blotting. *P<0.05 vs. mock treated HL-60 cells/vector. linc00239, long intergenic non-protein coding RNA 239; BEZ, NVP-BEZ235.

Effects of linc00239 on malignant behaviours, including proliferation, colony formation, migration and chemosensitivity, are abolished by NVP-BEZ235 treatment

As aforementioned (Fig. 5), activation of the PI3K/Akt/mTOR pathway by linc00239 was eliminated by NVP-BEZ235 treatment. To demonstrate whether linc00239 functioned via regulation of the PI3K/Akt/mTOR pathway and thus regulated malignant behaviours in AML cells, the effects of linc00239 on proliferation, colony formation, migration, and chemosensitivity were detected after the PI3K/Akt/mTOR pathway was inhibited by NVP-BEZ235 treatment in HL-60 cells. As anticipated, after NVP-BEZ235 pretreatment, the regulatory effects of linc00239 on the proliferation, colony formation, migration and chemosensitivity to doxorubicin were eliminated (Fig. 6). All of these data indicated that linc00239 regulated malignant behaviours mainly via activation of the PI3K/Akt/mTOR pathway.

Figure 6. linc00239 promotes malignant behaviours mainly via activating the PI3K/Akt/mTOR pathway. After 0.5 µM NVP-BEZ235 treatment, proliferation (A), colony formation (B), and migration (C) were detected. (D) Vector or linc00239-expressing vector-transfected HL-60 cells were pre-treated with 1 µM BEZ for 24 h and were detected by the CCK-8 assay to measure chemosensitivity. *P<0.05 vs. mock treated HL-60/vector. (E) CFSE/PI double staining was performed to detect the cell death rate after 2.68 µM doxorubicin treatment. *P<0.05 vs. mock treated HL-60/vector. (F) Annexin V-FITC/PI double-staining was performed to detect the apoptotic cell death rate after 2.68 µM doxorubicin treatment. *P<0.05 vs. mock treated HL-60/vector. linc00239, long intergenic non-protein coding RNA 239; BEZ, NVP-BEZ235; CCK-8, Cell Counting Kit-8; carboxyfluorescein diacetate succinimidyl ester; PI, propidium iodide.

Discussion

In the present study, by performing RT-qPCR and northern blotting in the AML cell lines KG-1 and HL-60, we revealed the existence of linc00239 RNA in these two cell lines was revealed, which was an expected result. In order to compare the relative amount of linc00239 in these two cell lines, RT-qPCR was performed, and the data were normalized to β-actin mRNA, GAPDH mRNA, or snoU6 RNA. These results indicated that the endogenous linc00239 level in KG-1 cells was much higher than that of HL-60 cells. The expression difference between these cell lines allowed us to investigate the roles of linc00239 in the malignant behaviours of AML. To achieve this purpose, KG-1 cells were used to downregulate linc00239; HL-60 cells were used to overexpress linc00239. In this way, it was revealed that the presence of linc00239 in AML cells was positively related to malignant behaviours, including proliferation, colony formation and migration.

Doxorubicin, a cytotoxic antiproliferative and apoptosis-inducing drug, was clinically and widely used as a cytotoxic agent for chemotherapy in several types of cancers, including AML (33,34). It disturbs the DNA replication processes, resulting in cell cycle arrest and cellular apoptosis (35). Thus, the effects of linc00239 on doxorubicin-induced cell cycle arrest and apoptotic cell death were investigated. Notably, without an evident effect on cell cycle arrest (data not shown), the presence of linc00239 significantly desensitized KG-1 and HL-60 cells to doxorubicin. To confirm the effects of linc00239 on chemosensitivity to doxorubicin, the apoptotic rate and the rate of cell death were evaluated, and the expected results were obtained. No obvious effects of linc00239 on cell cycle distribution were due to the induced cell death. Moreover, the existence of linc00239 also affected doxorubicin-induced cell death in an unknown manner.

It was also revealed that the presence of linc00239 was tightly related to the phosphorylation of AKT at S473 and mTOR at S2448, without affecting total protein levels. After pretreatment with an inhibitor of the PI3K/AKT/mTOR pathway, BEZ235, the presence of linc00239 failed to affect the PI3K/AKT/mTOR pathway and to exert protective effects against doxorubicin treatment. Moreover, when pre-treated with BEZ235, the regulatory roles of linc00239 on malignant behaviours in KG-1 or HL-60 cells were eliminated. PI3K/AKT/mTOR is considered as the most important factor in initiation and maintenance of AML, and the abnormality of this pathway was found in most AML cases (36). Our data revealed that the presence of linc00239 tightly affected the activation of the PI3K/ATK/mTOR pathway, indicating that it is a potential therapeutic target in AML. Moreover, in a future investigation, it is worth studying the epigenetic regulating role of microRNA and long non-coding RNA on this pathway.

In summary, it was revealed that linc00239 tightly regulated the activation of the PI3K/AKT/mTOR pathway without knowing the target in the PI3K/AKT/mTOR pathway, and by depending on this, it desensitized KG-1 or HL-60 cells to the chemoagent. The presence of linc00239 positively regulated the malignant behaviours, including proliferation, colony formation and migration in KG-1 and HL-60 cells. Collectively, linc00239 functioned as a PI3K/AKT/mTOR pathway activator and may be considered as a therapeutic target for AML treatment.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Chengdu Scientific Benefiting Project (no. 2015-HM01-00461-SF).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

YY performed the cellular and molecular experiments. WD contributed to the molecular experiments and was responsible for the data collection. YS performed the cellular experiments. ZZ performed the molecular experiments and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors state that they have no competing interests.

References