The leukemia model cell line K562 (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was cultured in Roswell Park Memorial Institute-1640 medium (Sigma-Aldrich) supplemented with 2 mM glutamine (Sigma-Aldrich), 10% fetal bovine serum (HyClone FBS; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Beyotime Institute of Biotechnology, Shanghai, China). The cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. The medium was changed weekly and cells were plated at a density of 1×10⁵ cells/ml. To explore the correlation between phosphorylated STAT3 (p-STAT3) and SCLIP, cells (1×10⁶) were seeded onto 6-well plates. With different treatment, cells were divided into three groups: Cells without treatment, cells treated with JSI-124 (1 µM; La Jolla, CA, USA) for 24 h and cells treated with recombinant interleukin-21 (10 ng/ml; Sigma-Aldrich) for 24 h.

Human SCLIP-specific double-strand siRNA (SCLIP-siRNA) and the negative control siRNA were purchased from Ambion (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell transfection was performed using Lipofectamine^® RNAiMAX Reagent (Ambion) according to the manufacturer's protocol. Prior to transfection, the K562 cells were cultured and seeded to become 60% confluent in 96-well plates. The SCLIP-siRNA (1 pmol for the SCLIP-siRNA group) or the negative control siRNA (1 pmol for the siRNA control group) was diluted and added to the corresponding wells. The cells were then incubated for 1 day at room temperature. The transfection efficiency was determined using a fluorescein-labeled siRNA. Untransfected cells served as the control group.

Cell proliferation and viability assays were conducted with Cell Proliferation kit I (Roche Life Science, Branford, CT, USA) according to the manufacturer's protocol. Specifically, cells were added to 96-well plates at a concentration of 2×10⁴ cells/ml and cultured for 3 days. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; 5 mg/ml) diluted in phosphate-buffered saline (PBS) was added to each well and incubated with the cells for 4 h. The cells were then centrifuged at 80 × g for 10 min at 4°C, collected and resuspended in dimethylsulfoxide. The plates were shaken until the crystals dissolved. The absorbance was determined at 490 nm using a spectrophotometer at 0 h, 72 h and 7 days after transfection. All assays were performed in triplicate.

Cell apoptosis assays were conducted by flow cytometry (FCM) using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). An Annexin V-FITC/PI Apoptosis Detection kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used and the experiment was performed according to the manufacturer's protocol. Specifically, the cell concentration was adjusted to 5×10⁵ cells/ml, and the cells were centrifuged at 200 × g for 5 min at room temperature and collected. After washing with cold PBS 3 times, the cells were resuspended in 200 µl 1X Binding Buffer, 10 µl Annexin V-FITC and 10 µl PI and then incubated in the dark for 15 min at room temperature. Prior to detection by FCM, another 300 µl 1X Binding Buffer was added. Detection was performed at 0 h, 72 h and 7 days after transfection. The Annexin V-positive and PI negative cells located in quadrant 3 (Q3) of the plot were considered to be apoptotic cells and were used for comparison among groups.

The expression levels of various mRNAs were detected by RT-qPCR using the following primers: STAT3, forward: 5′-GCCAGAGAGCCAGGAGCA-3′ and reverse: 5′-TGAAGCTGACCCAGGTAGCGCTGC-3′; SCLIP, forward: 5′-AGGAGTTATCTGTGCTGTCGC-3′ and reverse: 5′-TGGTAGATGGTGTTCGGGTG-3′; Bcl-2, forward: 5′-AAGAGCAGACGGATGGAAAAAGG-3′ and reverse: 5′-GGGCAAAGAAATGCAAGTGAATG-3′; and cyclin E1, forward: 5′-GCAAGCCTCGGATTATTGCA-3′ and reverse: 5′-CCTCTCTATTTGCCCAGCTCAGTA-3′. GAPDH was used as the internal control, with the following primer sequences: Forward: 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse: 5′-GACAAGCTTCCCGTTCTCAG-3′ The total RNA of cells was isolated with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) as well as DNase I (Promega Corporation, Madison, WI, USA) and complementary DNA synthesis was performed using a PrimerScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. qPCR was conducted in a 20-µl reaction system using a LightCycler 96 Real-Time PCR System (Roche Life Science, Basel, Switzerland) with the following cycling procedures: Pre-denaturation at 94°C for 2 min, followed by 40 cycles comprising denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min, and then a final extension step at 72°C for 7 min. All the experiments were performed in triplicate. The data were calculated using the 2^−ΔΔCq method (17).

The cells were harvested and washed twice with cold PBS. Protein samples were extracted with cell lysis buffer containing 2% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 100 µg/ml phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin. Equal amounts of protein (20 µg) from each group were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The blot was blocked with 5% skimmed milk in Tris-buffered saline Tween-20 (TBST) buffer (pH 8.0) overnight at 4°C and then incubated with specific primary antibodies for phosphorylated (p)-STAT3 (1:500; cat. no. sc-135649), SCLIP (1:500; cat. no. sc-85907) or β-actin (1:1,000; cat. no. sc-130656) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. The membrane was then washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-rabit IgG (1:5,000; cat. no. sc-2004) and donkey anti-goat IgG (1:5,000; cat. no. sc-2020) secondary antibodies (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Positive signals were detected using an enhanced chemiluminescence reagent (Amersham ECL Detection kit; GE Healthcare Life Sciences) and then analyzed using a Kodak Digital Imaging System (DC120; Kodak, Rochester, NY, USA). β-actin was used as the internal reference.

Differences between groups were examined using the unpaired Student's t-test. Data are presented as the mean ± standard deviation of three independent experiements. P<0.05 was considered to indicate a statistically significant difference. All data analyses were performed using Statistical Package for Social Sciences (SPSS) version 19 software (IBM SPSS, Armonk, NY, USA).

The phosphorylation levels of STAT3 and the protein expression levels of SCLIP were detected by western blotting. The results showed a positive correlation between the expression of phosphorylated STAT3 (p-STAT3) and SCLIP (Fig. 1A and B): When the phosphorylation of STAT3 was promoted by IL-21, the expression of SCLIP was upregulated and when the phosphorylation of STAT3 was inhibited by JSI-124, the expression of SCLIP was downregulated. The differences in the expression levels of SCLIP in the two treatment groups compared with those in the control group were significant (P<0.05). Since the expression of SCLIP could be activated by p-STAT3, these results imply that SCLIP might be a downstream molecule of STAT3.

Following the transfection of the cells with the SCLIP-specific siRNA or negative control siRNA, the mRNA expression levels of SCLIP and STAT3 in each group were detected by RT-qPCR. The expression levels of SCLIP and STAT3 mRNA in the siRNA control group were similar to those in the untransfected control group (Fig. 2). In the SCLIP-siRNA group, the expression of SCLIP mRNA was significantly downregulated (P<0.05) compared with that in the control group indicating that effective knockdown of SCLIP. However, the expression of STAT3 mRNA was hardly changed as SCLIP was inhibited, which implies that STAT3 is located upstream of SCLIP. These results together with the previous western blotting results, indicate that SCLIP may be upregulated by the phosphorylation of its upstream molecule, STAT3.

The effects of SCLIP on K562 cell viability and apoptosis were tested by MTT and cell apoptosis assays, respectively. The cell viability of the SCLIP-siRNA group was inhibited and exhibited a significant difference from the control group at 7 days after transfection (P<0.05; Fig. 3). Similarly, the apoptotic cell ratio in the SCLIP-siRNA group was significantly increased at both 72 h and 7 days after transfection compared with that in the control group (P<0.05), increasing from 4.28 to 5.85% and from 4.88 to 7.14%, respectively (Fig. 4). These data indicate that SCLIP was involved in the promotion of cell viability and inhibition of cell apoptosis in the K562 cells, implicating SCLIP as a regulator of the viability and apoptosis of CML cells.

The possible regulatory mechanisms of SCLIP in CML were studied by analyzing the changes in the expression of two anti-apoptosis genes, namely, Bcl-2 and cyclin E1, following the knockdown of SCLIP. The RT-qPCR results showed that the expression patterns of the two genes were positively associated with the expression of SCLIP (Fig. 5). That is, with the knockdown of SCLIP, both Bcl-2 and cyclin E1 mRNA expression levels were downregulated, exhibiting significant differences compared with the control group (P<0.05). Therefore, Bcl-2 and cyclin E1 are two possible downstream factors of SCLIP. It may be hypothesized that the two factors, together with STAT3 and SCLIP, constitute a part of the signaling pathway that regulates the viability and apoptosis of CML cells.