The THP-1 cells (derived from a patient with monocytic leukemia) and HL-60 cells (derived from a patient with promyelocytic leukemia) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The K562 cells (derived from a patient with chronic myelogenous leukemia) were obtained from CHI Scientific (Jiangyin, China). The U937 cells (derived from a patient with histiocytic lymphoma) were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The HL-60 cells were cultured in Iscove's modified Dulbecco's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit Haemek, Israel), 100 U/ml penicillin and 0.1 mg/ml streptomycin. The THP-1, K562, and U937 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) containing 10% FBS (Biological Industries), 100 U/ml penicillin and 0.1 mg/ml streptomycin. All cell lines were maintained in an incubator (Shanghai Lishen, Shanghai, China) with 5% CO₂ at 37°C.

Lentiviruses containing two short hairpin RNA (shRNA) sequences specific to ENDOCAN and a negative control (NC) were generated by a lentiviral packaging system from Wanleibio Co., Ltd. (Shenyang, China). The sequences were as follows: ENDOCAN shRNA1, 5′-TGGTGAAGAGTTTGGTATCTTTCAAGAGAAGATACCAAACTCTTCACCTTTTTTC-3′; ENDOCAN shRNA2, 5′-TCATCTGGAGATGGCAATATTTCAAGAGAATATTGCCATCTCCAGATGTTTTTTC-3′; NC shRNA, 5′-TTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTC-3′. The shRNA sequences were cloned into the AgeI/EcoRI sites of the lentiviral vector (LV-shRNA). The recombinant lentiviral vector and packaging plasmids were co-transfected into 293T cells (Procell Life Science and Technology Co., Ltd., Wuhan, China) with Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) and the supernatant containing the virus was collected to infect target cells.

Total RNA was extracted from cells using an RNApure High-purity Total RNA Rapid Extraction kit (BioTeke Corporation, Beijing, China) and reverse transcribed into cDNA using Super M-MLV reverse transcriptase (BioTeke Corporation). The cDNA was amplified by RT-qPCR using Exicycler™ 96 (Bioneer Corporation, Daejeon, Korea) with the following primers: ENDOCAN forward 5′-CTGGGAAACATGAAGAGCG-3′ and 5′-GCCTGAGACTGTGCGGTAG-3′ reverse; β-actin forward 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ and 5′-CTGTCACCTTCACCGTTCCAGTTT-3′ reverse. The reactions were performed in a 20-µl reaction mixture containing 1 µl template cDNA, 10 µl SYBR GREEN mastermix (2X), 0.5 µl forward and reverse primers and 8 µl ddH₂O. For the negative control, no primers were added. The thermocycling steps were as follows: Initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 10 sec, annealing at 60°C for 20 sec and elongation at 72°C for 30 sec, and a final extension at 72°C for 2 min 30 sec. Gene expression was analyzed using the 2^−ΔΔCq method (18).

Total protein was separated from cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with 1% phenylmethylsulfonyl fluoride. The nuclear protein was extracted using a nuclear protein extraction kit (Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA protein quantitative kit (Beyotime Institute of Biotechnology). Protein samples (20–40 µg/lane) were resolved by 8–15% SDS-PAGE, blotted onto polyvinylidene fluoride membranes, blocked with 5% skim milk and incubated with primary antibodies against ENDOCAN (1:300; cat. no. PAC463Hu01; Cloud-Clone Corp., Wuhan, China), proliferating cell nuclear antigen (PCNA; 1:500; cat. no. 10205-2-AP; Wuhan Sanying Biotechnology, Wuhan, China), cyclin D1 (1:500; cat. no. AP2612d; Abgent, Inc., San Diego, CA, USA), B-cell lymphoma 2 (BCL2; 1:500; cat. no. D160117; Sangon Biotech Co., Ltd., Shanghai, China), BCL-2-associated X protein (BAX; 1:1,000; D120073; Sangon Biotech Co., Ltd.), cleaved caspase 3 (1:1,000; cat. no. ab2302; Abcam, Cambridge, MA, USA), cleaved caspase 9 (1:1,000; cat. no. 9501; CST Biological Reagents Co., Ltd., Shanghai, China), cleaved poly (ADP-ribose) polymerase (PARP; 1:500; cat. no. ab32561; Abcam), NF-κB, P65 (1:1,000; cat. no. 10745-1-AP; Wuhan Sanying Biotechnology), phosphorylated (p-)P65 (1:400; cat. no. BM3994; Boster Biological Technology, Pleasanton, CA, USA), NF-κB inhibitor α (IκBα) (1:500; cat. no. bs-1287R; BIOSS, Beijing, China), p-IκBα (1:500; cat. no. bs-2513R; BIOSS), β-actin (1:500; bsm-33139M; BIOSS), and histone H3 (1:5,000; cat. no. GTX122148; GeneTex, Inc., Irvine, CA, USA) overnight at 4°C. Subsequently, the membranes were treated with horseradish peroxidase (HRP)-labeled goat anti-rabbit antibodies (1:5,000; cat. no. A0208; Beyotime Institute of Biotechnology) at 37°C for 45 min. The protein was visualized using enhanced chemiluminescence (ECL; Beyotime Institute of Biotechnology) with a gel imaging system (Beijing Liuyi Instrument Factory, Beijing, China). The protein expression levels were evaluated by densitometric analysis with Gel-Pro Analyzer 3.1 (Meyer Instruments, Inc., Houston, TX, USA) and each target protein was normalized to the corresponding β-actin or histone H3 band.

Cell proliferation ability was assessed using a CCK-8 assay (Wanleibio, Shenyang, China), based on the activity of mitochondrial dehydrogenases (19). Briefly, the cells were seeded into 96-well plates (3×10³ cells/well). The cells were cultured for 0, 24, 48, 72 and 96 h at 37°C with 5% CO₂. The supernatant was removed at each time point and replaced with 100 µl of complete medium, and 10 µl CCK-8 solution was added into each well for 1 h. The absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The cell cycle distribution was detected using a propidium iodide (PI)-based cell cycle analysis kit (Beyotime Institute of Biotechnology), as previously described (20). The principle of this method is that the nuclei of dead cells can be stained by PI. Briefly, the cells were collected and fixed in pre-cooled 70% ethanol for 2 h. The cells were then suspended in 500 µl of binding buffer containing 25 µl PI and 10 µl ribonuclease A (which can degrade RNA and prevent the PI staining of RNA) for 30 min at 37°C in the dark. The cell cycle was analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), based on forward light scatter, side light scatter and PI (FL-2 channel, wavelength 575 nm) fluorescence.

Cell apoptosis was evaluated using an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (Wanleibio Co., Ltd.), as previously described (21). Annexin V binds to the externalized phosphatidylserine of cells undergoing programmed cell death, and the nuclei of dead cells can be stained by PI. Briefly, the cells were collected and resuspended in 500 µl binding buffer. Subsequently, 5 µl Annexin V-FITC and 10 µl PI were added and mixed well for 15 min at room temperature away from light. The cells were analyzed using a flow cytometer (BD Biosciences), based on forward light scatter, side light scatter, and FITC (FL-1 channel, wavelength 530 nm) and PI (FL-2 channel, wavelength 575 nm) fluorescence.

The principle of EMSA is that DNA-protein complexes migrate at a slower rate than unbound DNA in a gel. The DNA binding activity of NF-κB was determined using the EMSA kit (Viagene Biotech, Ningbo, China), according to the manufacturer's protocol (22). Briefly, nuclear protein was extracted using a nuclear extraction kit (Beyotime Institute of Biotechnology). Protein concentration was determined using the BCA method. The nuclear protein (15 µg) was mixed with a biotin-labeled NF-κB probe for 20 min at room temperature. Subsequently, the DNA-protein complex was separated using a 6.5% polyacrylamide gel at 180 V for 80 min, transferred onto a nylon membrane at 360 mA for 1 h and crosslinked to the nylon membrane under UV light for 30 min. The bands were visualized using streptavidin-HRP and an ECL kit. The sequence of the probe used in EMSA was 5′-AGTTGAGGGGACTTTCCCAGGC-3′.

The results are presented as the mean ± standard deviation. Each experiment was repeated three times. Statistical differences were analyzed using Student's t-test, one-way or two-way analysis of variance or a Kruskal-Wallis test using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of ENDOCAN in leukemia cells, its expression was determined in four myeloid leukemia cell lines. The results of the RT-qPCR and western blot analyses showed that the expression of ENDOCAN was higher in the U937 and K562 cells than in the THP-1 and HL-60 cells (Fig. 1A and B). The U937 and K562 cells were then infected with lentivirus carrying ENDOCAN shRNA-1, −2 or scramble shRNA for 48 h. The mRNA and protein expression levels of ENDOCAN were evaluated by RT-qPCR and western blot analyses. As shown in Fig. 1C and D, its expression was significantly reduced in these cells in response to the ENDOCAN shRNAs.

Subsequently, experiments were performed to determine whether ENDOCAN knockdown affected cell proliferation. The U937 and K562 cells were treated with lentivirus harboring ENDOCAN shRNA-1, −2 or scramble shRNA for 0, 24, 48, 72 and 96 h. A CCK-8 assay was then used to evaluate the proliferation ability of the U937 and K562 cells. As shown in Fig. 2A, the proliferation potential of the ENDOCAN-silenced cells was distinctly inhibited. Furthermore, reduced expression of PCNA was identified in these cells following ENDOCAN silencing for 48 h, as determined by western blot analysis (Fig. 2B). In addition, following infection for 48 h, the flow cytometry results showed that ENDOCAN knockdown led to G0/G1 cell cycle arrest in the U937 and K562 cells (Fig. 3A). The western blot analysis results showed that the expression level of cyclin D1 was decreased in the ENDOCAN-silenced cells (Fig. 3B).

The apoptotic rates of ENDOCAN-silenced myeloid leukemia cells were then evaluated by flow cytometry using Annexin V/PI staining. Following infection of the U937 and K562 cells with lentivirus harboring ENDOCAN shRNA-1, −2 or scramble shRNA for 72 h, the apoptotic rate was significantly increased in the cells with ENDOCAN knockdown (Fig. 4A). In addition, the expression of apoptosis-related genes was detected by western blot analysis following infection for 72 h. As shown in Fig. 4B, the expression of BCL2 was decreased, whereas the levels of BAX, cleaved caspases 3 and 9, and cleaved PARP were increased in the ENDOCAN-silenced myeloid leukemia cells.

Finally, the effect of ENDOCAN knockdown on the activity of NF-κB was assessed in U937 and K562 cells infected with lentivirus carrying ENDOCAN shRNA-1, −2 or scramble shRNA for 48 h. As shown in Fig. 5A, the results of the western blot analysis showed that the expression of IκBα was increased, whereas the expression levels of p-IκBα and p-p65 were decreased following ENDOCAN knockdown in U937 and K562 cells. In addition, the expression of nuclear p65 was found to be reduced in the ENDOCAN-silenced myeloid leukemia cells, as determined by western blot analysis (Fig. 5B). The EMSA assay revealed that the DNA binding activity of NF-κB was attenuated in the myeloid leukemia cells following ENDOCAN silencing (Fig. 5C).