This study was approved by the Ethics Committee of San Er Ling Yi Hospital Affiliated to School of Medicine, Xi'an Jiaotong University, and each patient provided written informed consent. A total of 36 consecutive, treatment naive patients who were diagnosed with CML in our hospital and 8 healthy volunteers were enrolled in our study. Peripheral blood was then obtained from CML patients and healthy volunteers and the serum was collected by centrifugation at 1,500 g for 10 min at 4°C. Finally, serum were portioned in aliquots and stored at 80°C until subsequently use.

Human CML cell line K562 (American Type Culture Collection, Manassas, VA, USA) was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a 5% CO₂ humidified incubator at 37°C.

The miR-574-3p mimics, miR-574-3p inhibitor or their corresponding controls were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Vector pcDNA-IL6 was constructed by inserting the coding oligonucleotides of IL6 into pcDNA3.1 vector (Invitrogen, Shanghai, China) for overexpression of IL6. Small interference RNAs (siRNAs) targeting IL6 (si-IL6) and corresponding control siRNAs (si-control) were designed and synthesized by RiboBio (Gungdong, China). For cell transfection, K562 cells (6×10⁴ cells per well) were seeded in a 24-well plate and then transfected with 50 nM of miR-574-3p mimics or mimic control, miR-574-3p inhibitor or inhibitor control, pcDNA-IL6 or blank vector, and si-IL6 or si-control using Lipofectamine 2000 (Invitrogen, Shanghai, China) in accordance with the manufacturer's protocols. After 48 h of transfection, cells were harvested for subsequent analysis.

Complementary oligonucleotides containing the miR-574-3p target site from IL6 (IL6 3′UTR-wt) and the mutated 3′UTR of IL6 (IL6 3′UTR-mut) containing a mutated miR-574-3p target sequence with identical flanking nucleotides were synthesized by Invitrogen (Shanghai, China). Oligonucleotides were then inserted into the 3′UTR of the pMIR-REPORT luciferase reporter vector (Ambion, Austin, Texas, USA) to construct a luciferase reporter. pMIR-REPORT Beta-gal was used as the internal control. K562 cells were cultured into a 24-well plate and then cotransfected with miR-574-3p mimic or mimic control and pMIR 3′UTR clones or pMIR Beta-gal for 24 h using Lipofectamine 2000 (Invitrogen, Shanghai, China). The luciferase and b-galactosidase activity were then quantified using Dual-Light System chemiluminescent reporter gene assay (Applied Biosystems, Foster City, CA, USA).

The cell viability of K562 cells were detected by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-liumbromide (MTT) assay. After transfection, 6×10⁴ cells were plated into 6-well plates. At 1, 2, 3 days after transfection, 10 mg/ml of MTT solution (Sigma, USA) was added into each well and cultured the cells for 12 h. Then 200 µl of dimethyl sulfoxide (DMSO, Sigma, USA) was added into each well for 20 min to dissolve the formazan crystals. The absorbance at 490 nm was finally detected.

The colony formation ability of K562 cells was also detected. K562 cells in each group were diluted to 100 cells, seeded in soft agar plates and cultured in complete medium for 12 days. When the cells in the largest dilution formed clones, clones were then fixed with 100% methanol for 15 min. After that, cells were stained with Giemsa (Sigma, USA) for 15 min. The number of clones in 5 regions in each plate which were randomly selected was counted under a microscope (IX83, Olympus). Three parallel experiments were conducted for each sample.

The apoptosis of K562 cells was measured using Annexin V labeling BD Annexin V-fluorescein isothiocyanate (FITC) assay kit (Becton Dickinson, NJ, USA). In brief, a total of 5×10⁵ cells were harvested after transfection of 48 h. Cells were stained with 5 µl Annexin V-FITC and propidium iodide (PI) in the dark for 15 min. Within 1 h, the apoptotic cells were sent out by means of Flow BD Fac Canto II flow-cytometer (Becton Dickinson, NJ, USA). According to the fluorescence intensity, the apoptotic cells (annexin V-positive and PI-negative) were finally analyzed with a CellQuest 3.0 software (Becton Dickinson, NJ, USA).

Total RNA was extracted from peripheral blood and K562 cells using Trizol reagent (Invitrogen, Shanghai, China) and their quality was measured using ultraviolet spectrophotometer (SMA 400 UV-VIS, Merinton, Shanghai, China). First-strand cDNA was then synthesized using a High Capacity cDNA Reverse Transcription Kit (Invitrogen, Shanghai, China). To detect the expressions of miR-574-3p and IL6, qRT-PCR was performed using the SYBR ExScript RT-qPCR Kit (Takara, China) by means of the ABI PRISM 7300 Fast Real-Time PCR System (Ambion, Foster City, CA, USA). Moreover, analysis of melting curve was conducted at the end of each PCR to detect the specificity of the PCR products. U6 and Phosphoglyceraldehyde dehydrogenase (GAPDH) was used as the internal control for miR-574-3p and IL6, respectively. Finally, the relative expression of miR-574-3p and IL6 was calculated with 2^−ΔΔCt method.

Peripheral blood (2 ml) was then obtained from CML patients and healthy volunteers and the serum was collected by centrifugation at 350 g for 10 min. The concentration of IL6 was then determined with a commercially available ELISA kit (Genzyme, Boston, MA) according to the manufacturer's protocols.

Total proteins in whole-cell lysates of K562 cells were obtained using radioimmunoprecipitation buffer (RIPA) (Sangon Biotech, Shanghai, China) containing protease inhibitor cocktail (GE Healthcare, Buckinghamshire, UK) on ice for 20 min. After estimating the protein concentration by Bradford reagent (Biorad laboratories, CA, USA), equal quantities of protein extracts were separated in a 10% SDS-PAGE and then electrically transferred to polyvinylidene difluoride membrane (Amersham, GE, Buckinghamshire, UK). After blocked with 5% blocking agent, the membranes were immunoblotted with primary antibodies, including anti-IL6, anti-JAK1, anti-p-JAK1, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3 and anti-β-actin (1:1,000, cell signaling, MA, USA) overnight at 4°C in shaker. β-actin was used as the loading control. The membranes were then incubated with secondary horseradish peroxidase-conjugated antibody (1:5,000, cell signaling, MA, USA) for 1 h. Lastly, the membranes were visualized with Amersham ECL Detection Agent (GE, Buckinghamshire, UK) by exposure to hyperfilm (GE, Buckinghamshire, UK) in the dark room.

Data were presented as mean ± standard deviation (SD) and their normal distribution was conducted using one-sample K-S test. Significant differences between groups were assessed using Student's t-test. All statistical analyses were performed using SPSS 18.0 (SPSS, Chicago, IL) and P<0.05 was considered statistical significant.

As shown in Fig. 1A, the expression of miR-574-3p in peripheral blood obtained from CML patients was significantly lower than that in healthy controls (P<0.05), indicating miR-574-3p may be involved in the development and progression of CML. Overexpression of miR-574-3p inhibited proliferation and induced apoptosis of K562 cells.

To investigate the potential effects of miR-574-3p in CML, K562 cells were transfected with miR-574-3p mimic, mimic control, miR-574-3p inhibitor and inhibitor control. The results of MTT assay showed that after 2 and 3 days of transfection, miR-574-3p mimics significantly inhibited cell viability while miR-574-3p inhibitor markedly enhanced cell viability when compared with their corresponding controls (P<0.05, Fig. 1B). In comparison with their corresponding control groups, miR-574-3p expression was significantly increased in miR-574-3p mimic group, while obviously decreased in miR-574-3p inhibitor group after 2 days of transfection (P<0.05, Fig. 1C). Thus, colony formation assay and flow cytometry were further performed the cell proliferation and apoptosis after 2 days of transfection. The results showed miR-574-3p mimics decreased the number of colony (P<0.05, Fig. 1D) and induced cell apoptosis (P<0.05, Fig. 1E) significantly compared with mimic control. Moreover, miR-574-3p inhibitor resulted in opposite effects on colony number and cell apoptosis (Fig. 1D and E).

According to the information of TargetScanHuman, IL6 was the predicted as a potential target of miR-574-3p. Luciferase report assay was performed to further verify the predicted results. Expected results was obtained that miR-574-3p mimic significantly inhibited the luciferase report activity of IL6 3′UTR-wt rather than IL6 3′UTR-mut (P<0.05, Fig. 2A). Then qRT-PCR and western blot were performed to detect the expression of IL6 in different transfected groups. The results showed the expression of IL6 in both mRNA and protein levels was significantly inhibited in miR-574-3p mimic group compared with that in mimic control group, while was markedly increased in miR-574-3p inhibitor group compared with that in inhibitor control group (P<0.05, Fig. 2B and C). Notably, the mRNA expression of IL6 in peripheral blood obtained from CML patients and healthy controls was detected. Expected results were obtained that IL6 expression was significantly higher than that in healthy controls (P<0.05, Fig. 2D). ELISA assay also showed similar results that the concentration of IL6 was significantly higher than that in healthy controls (P<0.05, Fig. 2E). These data indicated that IL6 was the direct target of miR-574-3p and its expression was negatively regulated by miR-574-3p.

We further investigated whether dys-regulation of IL6 could also regulate K562 cell proliferation and apoptosis. Thus, K562 cells were transfected with pcDNA-IL6, blank vector, si-IL6 and si-control. As shown in Fig. 3A, when compared with their corresponding controls, overexpression of IL6 could significantly increase K562 cell viability after 2 and 3 days of pcDNA-IL6 transfection, while knockdown of IL6 resulted in a significantly decrease of cell viability after 2 and 3 days of si-IL6 transfection (P<0.05). In addition, after 2 days of transfection, the protein expression of IL6 was significantly up-regulated in pcDNA-IL6 group and obvious down-regulated in si-IL6 group in comparison with their corresponding control groups (P<0.05, Fig. 3B). Besides, the results of colony formation assay and flow cytometry showed, after 2 days of pcDNA-IL6 transfection, the number of colony was significantly increased (P<0.05, Fig. 3C) and apoptotic cells were markedly decreased (P<0.05, Fig. 3D). However, opposite effects were observed after 2 days of si-IL6 transfection (Fig. 1D and E). These data indicated that overexpression of IL6 promoted proliferation and inhibited apoptosis of K562 cells, which was opposite with the function of miR-574-3p overexpression.

We further investigated the relationship between miR-574-3p and IL6/JAK/STAT3 pathway, aiming to elucidate the key mechanism of miR-574-3p in CMI development. The results showed that the protein levels of IL6, p-JAK1/JAK1, p-JAK2/JAK2 and p-STAT3/STAT3 in miR-574-3p mimic group were all significantly lower than that in mimic control group or untransfected group (P<0.05, Fig. 4), indicating that overexpression of miR-574-3p could inhibited the activation of JAK/STAT3 pathway. However, the protein levels of IL6, p-JAK1/JAK1, p-JAK2/JAK2 and p-STAT3/STAT3 were significantly increased in miR-574-3p mimic+pcDNA-IL6 group in comparison with that in miR-574-3p mimic group or miR-574-3p mimic+blank vector group (P<0.05, Fig. 4), indicating that overexpression of IL6 could reverse the inhibitory of miR-574-3p overexpression on the activation of IL6/JAK/STAT3 pathway, and miR-574-3p might inhibit the activation of JAK/STAT3 pathway in K562 cells via targeting IL6.