Leukemia can be divided into acute leukemia and chronic leukemia. Both can be further divided into lymphocytic leukemia and myeloid leukemia. In addition, acute myeloid leukemia can be divided into M0-M7 according to the FAB classification criteria (13). Therefore, leukemia is a complex hematological malignant tumor, and each subtype has its own unique features. In the present study, six leukemia cell lines were used, namely the Jurkat human T cell acute lymphoblastic leukemia cell line, the BALL-1 B cell acute lymphoblastic leukemia cell line, the NB4, HL-60 and THP-1 acute myeloid leukemia cell lines and the K562 chronic myeloid leukemia cell line. All cell lines were donated by the Shanghai Institute of Hematology (Shanghai, China). The cells were routinely cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator containing 5% CO₂ at 37°C, as previously described (14). SC79 was provided by Selleck Chemicals.

Primary bone marrow samples were obtained from the inpatient and outpatient departments of Xiangya Hospital of Central South University (Changsha, China). A total of 36 patients with acute leukemia and 30 normal controls (healthy individuals without hematological conditions or other solid tumors) were included for tissue collection between 2016 and 2018; the patient characteristics are presented in Table I. Diagnosis was performed according to the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology: Acute lymphoblastic/myeloid leukemia (version 1.2015, http://www.nccn.org). Clinical data were collected from medical clinical records. The present study was approved by the Ethics Committee of Xiangya Hospital (approval no. 201603063).

Lentiviral vector construction was performed as previously reported (15). Briefly, short hairpin RNA (shRNA) against the human TAF-Iβ gene [shRNA-knockdown (KD)] and scramble shRNA, which acts as a negative control (shRNA-NC), were constructed by Shanghai GeneChem Co., Ltd. (Shanghai, China). The leukemic cells were then transfected with the shRNA-KD or shRNA-NC vectors using Lipofectamine^® 2000. The green fluorescent protein (GFP)-positive cells were counted under a fluorescence microscope. The RNA interference efficiency was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses.

Total RNA was extracted using an RNAfast200 kit (Fastagen, Shanghai, China). RT was performed with a One Step SYBR PrimeScript™ RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). The thermocycling conditions were as follows: 52°C for 5 min, 95°C for 10 sec. The primers sequences were as follows: TAF-Iβ, forward 5′-AAATATAACAAACCTCCGCCAACC-3′, reverse, 5′-CAGTGCCTCTTCATCTTCCTC-3′ and GAPDH, forward 5′-TGCACCACCAATGCTTAG-3′ and reverse 5′-GGATGCAGGGATGATGTTC-3′. The thermocycling conditions were as follows: 95°C for 5 sec, 60°C for 30 sec and 40 cycles, 4°C for 30 min and end of the PCR reaction. GAPDH was used for normalization; expression levels were quantified via the 2^−ΔΔCq method (16).

The specific experimental process of western blotting was performed according to our previous study (14). Briefly, the cells were washed with pre-cooled PBS, following which protein (30 µg) was extracted with radioimmunoprecipitation assay lysis buffer via centrifugation (5,000 × g for 20 min at 4°C), and separated by 12% SDS-PAGE. Following electrophoresis, the proteins were transferred onto nitrocellulose membranes and incubated with primary antibodies at room temperature for 2 h and 4°C overnight. The antibodies used were as follows: Anti-TAF-Iβ (cat. no. ab181990, mouse monoclonal, 1:1,000; Abcam, Cambridge, MA, USA), anti-PP2A (cat. no. ab32141, rabbit monoclonal, 1:5,000; Abcam), phosphorylated-glycogen synthase kinase-3β (GSK-3β; Ser9; cat. no. 9336, rabbit monoclonal, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated-AKT (Ser473; cat. no. 9271, rabbit monoclonal, 1:2,000; Cell Signaling Technology, Inc.), anti-caspase-3 (cat. no. 9662), anti-poly(ADP-ribose)polymerase (PARP; cat. no. 9542, rabbit polyclonal, 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. sc-166574, mouse monoclonal, 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, the washed membranes were incubated with secondary antibodies (cat. no. sc-2005; goat anti-mouse or cat. no. sc-2004; goat anti-rabbit IgG, 1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature and visualized via chemiluminescence (Bio-Rad chemiluminescence imaging system; Bio-Rad Laboratories, Inc.).

Cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), which was performed according to the manufacturer's protocol. A total of 100 µl of cell suspension (5,000 cells/well) was inserted into a 96-well plate and the cells were cultured at 37°C in 5% CO₂ for 48 h. Following culture, 10 µl CCK-8 solution was applied to each well of the plate and cells were incubated for 3 h in an incubator at 37°C. Subsequently, the absorbance was measured at 450 nm using a multifunctional microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

This assay was performed using semisolid methylcellulose medium (Stemcell Technologies, Inc., Vancouver, BC, Canada) as previously described (17).

Each group of cells were washed with pre-cooled PBS three times, following which the cell cycle and apoptosis were analyzed according to the manufacturer's protocols of the cell cycle staining and Annexin V-phycoerythrin/7-aminoactinomysin D apoptosis kits (Multi Sciences, Hangzhou, China), respectively, with a BD FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). For cell cycle analysis, ~1×10⁶ cells were incubated with 1 ml DNA staining solution and 10 µl permeabilization solution. The cells were vortexed for 10 sec and incubated in the dark at room temperature for 30 min and analyzed. For the detection of apoptotic cells, 3×10⁶ untreated cells were resuspended with 500 µl apoptosis-positive control solution. Following incubation on ice for 30 min, the supernatant was discarded. Subsequently, 1.5 ml 1X binding buffer was added. This cell suspension was divided into three groups, labeled as the blank control, single staining A (5 µl Annexin-fluorescein isothiocyanate staining for 5 min without light) and single staining B (10 µl propidium iodide staining for 5 min without light) groups. Subsequently, the parameters for FCM (488 nm excitation and band pass filters of 530/30 nm for FITC detection and 585/42 nm for 7-AAD detection) were set for the analysis of the aforementioned cell groups. As the six leukemia cell lines all underwent the same treatment. The flow parameters of each cell line were not set separately. Although this may result in a high background state in certain cell lines, it does not cause a qualitative change in the final statistical results. Based on these criteria, all other analyses were performed.

All statistical evaluations were performed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA); analyses were repeated at least three times. Statistical significance was determined using Student's t-test or one-way analysis of variance (followed by the LSD post hoc test). P<0.05 was considered to indicate a statistically significant difference.

The expression of TAF-Iβ was examined in six typical leukemic cell lines and normal human bone marrow mesenchymal stem cells (HBMSCs). The western blot (Fig. 1A) and RT-qPCR (Fig. 1B) analyses revealed increased expression levels of TAF-Iβ in almost all leukemic cell lines compared with those in the HBMSCs. Additionally, the RT-qPCR analysis of clinical samples from 36 patients with acute leukemia and 30 normal controls demonstrated that 72.2% of the leukemic bone marrow specimens (26/36 samples) exhibited upregulated expression of TAF-Iβ. By contrast, the normal control specimens exhibited relatively low expression (Fig. 1C).

In order to determine whether TAF-Iβ serves a functional role in leukemic cells, shRNA-KD or shRNA-NC were transfected into the Jurkat, BALL-1, NB4, HL-60, THP-1 and K562 cells. The results demonstrated that the transfection efficiency (GFP-positive cell count) was >80% (Fig. 2A). In addition, the mRNA and protein expression levels of TAF-Iβ were significantly decreased in the KD groups compared with those in the CON groups (P<0.01; Fig. 2B and C).

To evaluate the effects of TAF-Iβ KD on leukemic cell proliferation in vitro, a CCK-8 assay was performed. The results revealed that, compared with those of the CON and NC groups, the proliferative abilities of cells in the KD groups were significantly inhibited (P<0.05; Fig. 3A). In accordance with these findings, TAF-Iβ KD in the cells led to decreases in the number and size of colonies (P<0.05; Fig. 3B). By contrast, analysis by FCM indicated that the percentages of S-phase Jurkat, BALL-1 and HL-60 cells in the KD groups were notably reduced compared with those in the CON groups; the numbers of G₀/G₁- or G₂/M-phase cells were significantly increased (P<0.01; Fig. 3C).

The results of the present study demonstrated that the apoptotic rates of the Jurkat, BALL-1, NB4, HL-60, THP-1 and K562 cells in the KD groups were 21.51±0.77, 27.33±0.77, 61.67±0.77, 35.70±0.77, 15.48±0.77 and 40.55±0.77%, respectively, which was notably higher than those in the corresponding control groups (Fig. 4A). Additionally, the expression levels of cleaved caspase-3 (p19) and PARP (p89) were significantly increased compared with those in the CON groups, indicating the induction of apoptosis (P<0.01; Fig. 4B).

TAF-Iβ was originally identified as a potent physiological inhibitor of PP2A; therefore, the expression levels of PP2A were determined following TAF-Iβ silencing. As expected, TAF-Iβ KD resulted in upregulated expression levels of PP2A (P<0.05; Fig. 5A). Additionally, as TAF-Iβ may be associated with the expression of GSK-3β in cancer and diseases of the central nervous system (18), alterations in the expression of GSK-3β were analyzed in NB4 and K562 cells. TAF-Iβ KD appeared to inhibit the phosphorylation of GSK-3β, as demonstrated by reductions in the levels of Ser9-phosphorylated GSK-3β (P<0.05; Fig. 5B). To further investigate the mechanism regulating the expression of GSK-3β, the expression of AKT was analyzed, which is a well-known kinase that is activated by various factors and is involved in the regulation of cell proliferation, differentiation, metastasis and apoptosis (19). The results demonstrated that TAF-Iβ KD markedly inhibited the expression of AKT; this effect was reversed by SC79, an inducer of AKT. Additionally, the TAF-Iβ KD-mediated downregulation of GSK-3β was suppressed following treatment with SC79 (P<0.01; Fig. 5C and D).