Cell lines and cultures

Human myeloid leukemia cell lines including K562, NB4, U937, HL-60 and imatinib-resistant K562/G01 and HEK 293T cells were selected from CCTCC (China Center for Type Culture Collection, Wuhan, China) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Myeloid leukemia cell line Kasumi-1 (kindly provided by Prof. Ligen Liu, the Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China) was cultured in RPMI-1640 medium supplemented with 15% FBS. K562/G01 cells grown in an imatinib-free culture medium (4 μM) for at least two weeks for each experiment. HEK 293T cell line was cultured in DMEM (Invitrogen) containing 1% penicillin/streptomycin and 10% FBS. All cell lines were incubated at 5% CO2 and 37℃.

Construction of miR-29b-3p over- and down-expression vectors

The lentivirus vector containing overexpressed and scrambled miR-29b-3p was constructed by GeneChem Company (Shanghai, China), the sequences of miR-29b-3p synthetic vectors as following: 5'-UAGCACCAUUUGAAAUCAGUGUU-3'. miR-29b-3p mimic, inhibitor and relevant negative controls were synthesized by GenePharma Company (Shanghai, China), the sequences of synthetic vectors as following: miR-29b-3p mimic: 5'-UAGCACCAUUUGAAAUCAGUGUU-3', miR-29b-3p inhibitor: 5'-AACACUGAUUUCAAAUGGUGCUA-3'.

Cell transfection

According to the lentivirus infection protocol, the optimal infection conditions of K562 and U937 cells respectively included the multiplicity of infection were 20 and 50, supplemented with enhancer infection supplement (ENI.S.) and polybrene. Stably infected cell lines were selected with 1.7 μg/ml puromycin (Sigma-Aldrich). Then the cells were divided into the miR-29b-3p group (transfected with a miR-29b-3p lentivirus vector), the NC group (transfected with a scramble-miR-29b-3p lentivirus vector) and the CON group (blank control). miR-29b-3p stably transfected cells were harvested at 96h post-infection for various assays subsequently reported in this study.

Electric transfection reagents containing cells transfected with miR-29b-3p mimics, inhibitor or negative controls using the TransEasy electrical transfection kit (Cellapy Biotechnology, China) according to the manufacturer’s instruction were added to electrode cups, which were placed in an X Unit (Lonza Nucleofector™4D, Switzerland) to switch on the procedure. The transfected cells were incubated at 5% CO2 and 37℃ for 48h.

Dual-luciferase reporter gene assay

HuR-3′UTR luciferase reporter plasmids with wild-type HuR-3′UTR (wt UTR) and mutated HuR-3′UTR (mut UTR) in the predicted miR-29b-3p binding site were constructed by GenePharma (Shanghai, China), the sequences of synthetic vectors as following: HuR wt: 5'-CTCTAGTCGCAGCTCTGTGACTGATTCCCTCCCGGGTGCTGAGTCCCCTCCCCGGCCACC-3',

HuR mut: 5'-CTCTAGTCGCAGCTCTGTGACTGATTCCCTCCCGCCACGAGAGTCCCCTCCCCGGCCACC-3'. The dual luciferase reporter plasmids with HuR wt UTR, or with mut UTR and miR-29b-3p or scramble mimics, which were co-transfected into HEK 293T cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacture’s instruction. After 48h of transfection, the firefly and renilla luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega, USA). The relative luciferase unit (RLU) activity was determined as follows, with the cell lysate of the reporter gene regarded as the blank control and the Renilla luciferase as the internal control: RLU = RLU firefly luciferase / RLU Renilla luciferase.

Real-time quantitative PCR analysis

Total RNAs were extracted using Trizol reagent according to the manufacturer’s protocol. The reverse transcriptase (RT) reactions of miR-29b-3p and HuR were respectively amplified using a Bluge-LoopTM miRNA qRT Starter Kit (RiboBio, Guangzhou) and a RevertAid First Strand cDNA Synthesis Kit (Thermo ScientificTM). Quantitative PCR (qPCR) analyses for miR-29b-3p and HuR were performed on a 7500 Real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA), using a Bluge-LoopTM miRNA qPCR Starter Kit (RiboBio, Guangzhou) and UltraSYBR Mixture (Low Rox), respectively. The primer sequences of HuR for qPCR were shown as flowing: Forward: 5'-GGCGCAGAGATTCAGGTTCT-3', Reverse: 5'-TCCTGCCCCAGGTTGTAGAT-3'. The relative quantification of miR-29b-3p and HuR expressions normalized to U6 or GAPDH was calculated using the comparative 2-ΔΔCt method.

Western blot analysis

Total protein was extracted from cells and lysed using RIPA buffer (Boster, USA), and the protein concentration was determined by the BCA Kit (Boster, USA). Nuclear and cytoplasmic extracts were prepared using the nuclear and cytoplasmic protein extraction kit (Cwbio, China) following the manufacturer’s protocol. Equal amounts from the cell lysates were separated by SDS-PAGE and transferred electrophoretically onto PVDF membranes. The membranes were cut prior to hybridisation with antibodies during blotting. After blocking, the membranes were incubated with the following specific primary antibodies overnight: anti-HuR (ab200342, Abcam), anti-p65 (ab32536,Abcam), anti-phospho-p65 (Ser536) (#3031S,CST), anti-IκBα(#9242S, CST), anti-phospho-IκBα (Ser32/36) (5A5) (#9246S, CST), anti-STAT1 (ab109320, Abcam), anti-phospho-STAT1(Y701) (ab30645,Abcam), anti-STAT3 (ab68153, Abcam), anti-phospho-STAT3 (Tyr705) (#8204, CST), anti-STAT5 (D3N2B) (#25656, CST), anti-phospho-STAT5 (Tyr694) (D47E4) (#4322, CST). Afterward, the membranes were incubated with the HRP-conjugated secondary antibody (SSA016, Sino biological) for 2h at room temperature. Solution A and solution B of ECL chemiluminescence kit were mixed in a ratio of 1:1 for one minute. The PVDF membrane was moved to the exposure table, and the chromogenic solution was dripping to cover the surface of the membrane, then put it into the Bio-Rad ChemiDoc XRS+ Chemiluminescence Imager for exposure. Protein bands were visualized and quantitated using the Image J 1.43 software (NIH, MD, USA), and data were normalized to GAPDH (#5174, CST) and PCNA (10205-2-AP, Proteintech).

MTS assay

Cells were seeded to 96-well plates and were detected at different times points (24h, 48h, 72h and 96h) using the MTS assay (Promega, USA). The absorbance was measured at 492/630 nm using a Microplate Reader (MK3, Thermo Fisher Scientific, USA).

Cell clone formation assay

The cells were cultured in 24-well plates in RPMI-1640 medium containing 1.6% methyl cellulose (Sigma, USA) for 7-10 days. The number of colonies (containing ≥ 40 cells) was counted and the efficiency of colony formation was assessed.

Flow cytometry

Cell apoptosis was examined using Annexin V-PE and 7-AAD staining assays (BD Bioscience, USA) following the manufacturer’s protocols, and cell cycle was analyzed using a Cell Cycle Analysis Kit (Keygen Biotech, China). Cell cycle and apoptosis were detected by flow cytometry (FCM) analysis (Accuri C6, BD Bioscience, USA).

Transwell assay

In-vitro invasion and migration were analyzed using 8 µm pore transwell chambers (Coring, MA, USA). The cells were reseeded onto the matrigel-coated upper chambers (Corning, USA) containing serum-free RPMI-1640, and 10% FBS was added to the lower chambers. Cells were incubated for 24h for invasion assay. For invasion assay, cells attaching to the lower surface of the membrane were fixed by methanol and stained with Wright-Giemsa, and the number of cells was counted under a light microscope (IX71, Olympus, Japan). For migration assay, cells migrating to the lower chambers were collected and detected using MTS.

Immunofluorescence assay

Cells were fixed with 4% paraformaldehyde (DingGuo, Beijing, China) for 15min at room temperature, and permeabilized with 0.1%Triton X-100 (Sigma, USA) for 15 min. After blocking with 5% goat serum (Gibco, USA) for 2h at room temperature, cells were incubated with anti-p65 antibody (at a dilution of 1:200, Affinity Biosciences, USA) overnight at 4℃. Then cells were incubated with a 1:500 dilution of Alexa Fluor 555 fluorescein-labelled goat anti-rabbit IgG antibody (Dingjie, China) in darkness for 2h. After the cells for washing three times and stained with DAPI (Servicebio, Wuhan, Chian) for 5 min, images were captured by a camera on an inverted fluorescence microscope (Olympus, Japan).

HuR knockdown and rescue experiment

To silence HuR expression, a small interfering RNA (siRNA) targeting HuR (5'-AAGAGGCAAUUACCAGUUUCA-3') and a control siRNA (5'-UUGUUCGAA CGUGUCACGUTT-3') were purchased from GenePharma Company (Shanghai, China). Three groups were established, which included HuR-KD group (transfected with HuR siRNA), HuR-NC group (transfected with control siRNA) and rescue group (co-transfected with HuR siRNA and miR-29b-3p inhibitor). All transfections were performed using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s instructions. Then biological behaviors of AML cells including cell proliferation, clone formation, cell cycle and apoptosis, invasion and migration were carried out according to the above steps.

Statistical analysis

All experiments were repeated at least three times. Data were expressed as mean± standard deviation (SD) using SPSS 24.0 statistical software. For variables with a normal distribution, comparisons between two groups and homogeneity of variance was verified using an independent samples t-test. Differences between multiple groups were compared using one-way ANOVA. For variables with a non-normal distribution, a nonparametric test was employed. Spearman’s coefficient was used for determining the correlation between two variables. A P value of <0.05 was considered statistically significant.