The ‘Boxplots’ module of the GEPIA database (gepia.cancer-pku.cn) was used to analyze the expression of RAB34 in the tissues of patients with AML and the ‘Survival Analysis’ module of the GEPIA database was to analyze the correlation between RAB34 and the overall survival rate of patients with AML (13). HumanTFDB (http://bioinfo.life.hust.edu.cn/) was used to predict the binding sites of E2F1 on the promoter of RAB34(14).

Human bone marrow stromal HS-5 cells (cat. no. BFN60808921) and AML MOLM-14 cells (cat. no. BFN60810333) were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd. AML MV4-11 (cat. no. CRL-9591) and HL-60 cells (cat. no. CCL-240) were purchased from the American Type Culture Collection. The AML Kasumi-1 cell line (cat. no. SCSP-5015) was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS (both from Thermo Fisher Scientific, Inc.) and 100 IU/ml penicillin + 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37˚C with 5% CO₂.

RNA samples were extracted from cells with chloroform and a TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed to cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (cat. no. R211-01; Vazyme Biotech Co., Ltd.). The conditions for reverse transcription were as follows: 37˚C for 10 min, followed by 85˚C for 5 sec and then storage at 4˚C. qPCR was subsequently performed using the HiScript II One Step qRT-PCR SYBR Green kit (Vazyme Biotech Co., Ltd.), 1 µl cDNA and amplification primers. The thermocycling conditions used were as follows: Denaturation at 95˚C for 30 sec, followed by 40 cycles at 95˚C for 5 sec and 60˚C for 30 sec. The following primer pairs sequences were designed by Guangzhou RiboBio Co., Ltd.: RAB34 forward, 5'-GTCTCCGATTCCCCATCACC-3' and reverse, 5'-ATGCGGACAACATCCCCAAT-3'; E2F1 forward, 5'-GGGGGAGAAGTCACGCTATG-3' and reverse, 5'-AAACATCGATCGGGCCTTGT-3' and GAPDH forward, 5'-CATGAGAAGTATGACAACAGCCT-3' and reverse, 5'-AGTCCTTCCACGATACCAAAGT-3'. The mRNA expression levels were quantified using the 2^-ΔΔCq method (15) and normalized to that of the internal reference gene GAPDH.

Total protein was extracted from the cells using RIPA lysis buffer (Cell Signaling Technology, Inc.) and quantified using a BCA kit (Beyotime Institute of Biotechnology). Total protein (30 µg/lane) was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% milk for 1 h at 37˚C and then incubated in the primary antibodies, against RAB34 (1:1,000; cat. no. PA5-99697; Thermo Fisher Scientific, Inc.), Bcl-2 (1:1,000; cat. no. ab32124; Abcam), Bax (1:1,000; cat. no. 182733; Abcam), CDK4 (1:1,000; cat. no. ab108357; Abcam), CDK8 (1:1,000; cat. no. ab229192; Abcam), cyclin D1 (1:1,000; cat. no. ab16663; Abcam), E2F1 (1:1,000; cat. no. ab288369; Abcam) or GAPDH (1:1,000; cat. no. ab9485; Abcam), overnight at 4˚C. On the next day, membranes were incubated with the HRP-conjugated secondary antibody (goat anti-rabbit; 1:5,000; cat. no. ab6721; Abcam) for 2 h at 37˚C. Finally, all the membranes were visualized using a BeyoECL Plus kit (Beyotime Institute of Biotechnology) on an Odyssey Infrared imaging system (Bio-Rad Laboratories, Inc.) and semi-quantified using ImageJ software (version 1.42; National Institutes of Health).

Lentivirus short hairpin (sh)RNA RAB34 sequences (shRNA-RAB34#1 and shRNA-RAB34#2) which were ligated into the plasmid of U6/GFP/Neo and scrambled control shRNA (shRNA-NC) were established and synthesized by Jimon Biotechnology (Shanghai) Co., Ltd. After 48 h of transfection, 2 µg/ml puromycin (Beyotime Institute of Biotechnology) was added to create stably transfected HL-60 cell lines at a multiplicity of infection of 10, followed by maintenance with 0.5 µg/ml puromycin. The E2F1 overexpressing lentivirus (Oe-E2F1) tagged with Flag (with the E2F1 gene inserted into the pcDNA3.1 vector), was purchased from Jimon Biotechnology (Shanghai) Co., Ltd. An empty vector served as the negative control (Oe-NC), and the interim cell line used was the 293T cell line which was purchased from the American Type Culture Collection. The 2nd generation system was used. ShRNAs (500 ng/µl) and pcDNA3.1 vectors (4 µg) were transfected into HL-60 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37˚C. The sequences of two human shRNA-RAB34 and the shRNA-NC were as follows: Sh-RAB34#1, 5'-CCGCGTAATCGTAGGAACTAT-3'; sh-RAB34#2, 5'-CGCGTAATCGTAGGAACTATC-3'; and shRNA-NC, 5'-CAACAAGATGAAGAGCACCAA-3'. After 48 h incubation at 37˚C, transfected cells were harvested and utilized for further experiments.

Cell viability was assessed using a CCK-8 assay kit (Beyotime Institute of Biotechnology) following the manufacturer's protocol. After 48 h of transfection, HL-60 cells were seeded into 96-well plates at the density of 1,000 cells/well. RPMI-1640 medium containing 10 µl CCK-8 was then added for 4 h. The absorbance in each well at 450 nm was then measured using a microplate reader (Bio-Rad Laboratories, Inc.).

EdU staining was used to analyze the cancer cell proliferation according to the following protocol. Briefly, HL-60 cells were transfected and then incubated with EdU (20 mmol/l; cat. no. KGA331-1000; Nanjing KeyGen Biotech, Co., Ltd.) at 37˚C for 2 h to infiltrate thymine into the DNA molecule being synthesized during DNA replication. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. DAPI (10 µmol) was used to stain the nucleus for 5 min at room temperature. Finally, the images were captured with a fluorescence microscope (Olympus Corporation; magnification, x200).

Flow cytometry was used to observe the cell cycle distribution. Briefly, HL-60 cells (4x10⁵) were collected after 48 h of transfection and were stained with 50 µg/ml PI (Dojindo Molecular Technologies, Inc.) for 15 min at room temperature. Cell cycle distribution and sub-G₁ DNA content were analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences) and the data were analyzed with ModFit 2.0 software (Verity Software House, Inc.).

Cell apoptosis was evaluated through flow cytometry using a cellular Annexin V-FITC/PI Kit [cat. no. 70-AP101-100; Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.] according to the manufacturer's protocol. Cells were plated into six-well plates at a density of 4x10⁵ cells/well. After 48 h of transfection, cells (4x10⁵ cells/well) were incubated with 200 µl binding buffer and double stained with Annexin V-FITC and PI at 4˚C in the dark for 20 min. The cells were then assessed using BD FACSCalibur™ (BD Biosciences) and the data were analyzed with ModFit 2.0 software (Verity Software House, Inc.).

The pGL3 plasmid (Promega Corporation) containing the RAB34 promoter region element was generated by site-directed mutagenesis and subcloned into the pGL3-basic luciferase reporter vector. A mutant type (MUT) and wild-type (WT) RAB34 promoter vector were produced by GeneCopoeia, Inc.. The Lipofectamine^® 2000 transfection reagent was used to co-transfect the HL-60 cells with 400 ng aforementioned plasmids and 100 nM Oe-E2F1 or the Oe-NC plasmids. After 48 h incubation at 37˚C, the luciferase activity was assayed using a Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocol. The relative luciferase activity was calculated by normalizing the luminescence intensity of the firefly luciferase activity to that of the Renilla luciferase activity.

The cells were treated with 4% formaldehyde for 10 min at room temperature to generate DNA-protein cross-links. A Bioruptor^® (Diagenode, Inc.) was applied to sonicate cell lysates (20 kHz; 4 pulses of 12 sec each, followed by 30 sec rest on ice between each pulse) to generate chromatin fragments with an average size of 500 bp. A total of 40 µl protein A/G agarose beads (cat. no. sc-2003; Santa Cruz Biotechnology, Inc.) was added to the lysates and the lysates (500 µg) were then immunoprecipitated for 6 h with 5 µg specific antibody against E2F1 (ab245308; Abcam) at 4˚C. Normal IgG antibody (ab172730; Abcam) was used as a control. The next day, 30 µl protein G agarose beads were added and the precipitate was collected after incubating at 4˚C for 6 h and centrifuged at 1,000 x g at 4˚C for 3 min. The precipitate was washed with 5X lysis buffer and resuspended in 150 µl 1X ChIP Elution Buffer. Chromatin from the beads were eluted with gentle vortexing (1,200 rpm) at 65˚C for 30 min. DNA was purified using the DNA Purification kit (cat. no. D0033; Beyotime Institute of Biotechnology). Relative enrichment was evaluated using RT-qPCR. The primers used were as follows: RAB34 forward, 5'-GTCTCCGATTCCCCATCACC-3' and reverse, 5'-ATGCGGACAACATCCCCAAT-3'.

Data were presented as mean ± standard deviation and analyzed using the one-way ANOVA with Tukey's post hoc test with GraphPad Prism 5.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference. Each test was repeated ≥ three times.

GEPIA database analysis revealed that RAB34 was highly expressed in the tissues of patients with AML compared with normal tissues (Fig. 1A), where higher RAB34 expression levels were significantly associated with poorer overall survival in patients with AML (Fig. 1B). RT-qPCR and western blotting showed that RAB34 expression was markedly increased in AML cell lines (Fig. 1C and D). Since the expression level of RAB34 was the highest in HL-60 cells, HL-60 cells were selected for subsequent experiments.

The RAB34 shRNA plasmid was constructed before the knockdown efficiency was detected by RT-qPCR and western blotting (Fig. 2A and B). Since the degree of knockdown efficiency in the shRNA-RAB34#2 group was higher, shRNA-RAB34#2 was chosen for subsequent experiments, which were divided into the control, shRNA-NC and shRNA-RAB34 groups. Cell proliferation was detected by CCK-8 assay and EdU staining, which showed that compared with that in the shRNA-NC group, cell proliferation in the shRNA-RAB34 group was markedly decreased (Fig. 2C and D).

Flow cytometry analysis of the cell cycle distribution showed that compared with that in the shRNA-NC group, the proportion of cells in the G₀/G₁ phase in the shRNA-RAB34 group was significantly increased, whilst that in the S phase was significantly decreased (Fig. 3A). The expression of cell cycle marker proteins CDK4, CDK6 and cyclin D1 was next detected by western blotting, which showed that knocking down RAB34 expression significantly inhibited the expression of CDK4, CDK6 and cyclin D1 (Fig. 3B). Flow cytometry and western blotting were subsequently used to analyze cell apoptosis and the results showed that RAB34 knockdown significantly promoted cell apoptosis (Fig. 3C). In addition, significant decreases in the expression of the apoptosis marker protein Bcl-2 and an significant increases in the expression of Bax were observed (Fig. 3D).

Based on the binding motif of E2F1, the HumanTFDB website predicted the potential binding for the transcription factor E2F1 on the RAB34 promoter (Fig. 4A). The expression of E2F1 in HL-60 cells was significantly increased compared with HS-5 cells as shown through RT-qPCR and western blotting results (Fig. 4B and C). Subsequently, the E2F1 overexpression plasmid was constructed and it was discovered than E2F1 expression was significantly elevated by transduction of Oe-E2F1 plasmids, which indicated successful transfection (Fig. 4D and E). Through the luciferase reporter assay, it was demonstrated that the luciferase activity of RAB34-WT was markedly enhanced after E2F1 was overexpressed (Fig. 4F). The ChIP assay also demonstrated that the RAB34 promoter was enriched in the E2F1 antibody (Fig. 4G). In addition, RT-qPCR and western blotting showed that RAB34 expression in HL-60 cells was significantly increased after overexpressing E2F1 (Fig. 4H and I).

The regulatory mechanism of RAB34 on the proliferation and apoptosis of AML cells was next investigated. Cells were divided into the control (shRNA-NC), shRNA-RAB34, shRNA-RAB34 + Oe-NC and shRNA-RAB34 + Oe-E2F1 groups. CCK-8 and EdU staining showed that compared with that in the shRNA-RAB34 + Oe-NC group, cell proliferation of the shRNA-RAB34 + OE-E2F1 group was markedly increased (Fig. 5A and B). Flow cytometry showed that compared with that in the shRNA-RAB34 + Oe-NC group, the proportion of cells in the G₀/G₁ phase in the shRNA-RAB34 + Oe-E2F1 group was significantly decreased, whilst that in the S-phase in the shRNA-RAB34 + Oe-E2F1 group was significantly increased (Fig. 5C). Western blotting showed that E2F1 overexpression significantly reversed the inhibitory effects of RAB34 knockdown on CDK4, CDK6 and cyclin D1 protein expression (Fig. 5D).

Subsequently, analysis of apoptosis showed that compared with that in the shRNA-RAB34 + Oe-NC group, the shRNA-RAB34 + Oe-E2F1 group showed a significant decrease in the levels of apoptosis (Fig. 6A). Furthermore, shRNA-RAB34 + Oe-E2F1 co-transfection significantly increased Bcl-2 expression whilst decreasing Bax expression, compared with those in the shRNA-RAB34 + Oe-NC group (Fig. 6B).