Cell lines and culture
HS-5 and AML cell lines ((HEL, NB4, HL-60, and THP-1) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) and incubated in a humidified atmosphere containing 5% CO2 at 37 °C.
Cell Transfection
To overexpress the expression of TNK2-AS1, the full sequence of TNK2-AS1 was introduced into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) to generate pcDNA-TNK2-AS1 constructs. Likewise, to silence the expression of TNK2-AS1, ELK1, EZH2 and CELF2, shRNAs specifically targeting them were synthesized and obtained from Genepharma (Shanghai, China). The above negative controls (vector and sh-NC) were also synthesized by Genepharma (Shanghai, China). Lipofectamine 2000 from Invitrogen was utilized to transfect above in cells.
Quantitative real-time PCR (RT-qPCR)
Total RNAs were extracted by using Trizol (Invirtogen, Carlsbad, CA, USA). The cDNA was synthesized from total RNA by using Prime Script RT reagent (TaKaRa, Tokyo, Japan). Quantitative real time-PCR (RT-qPCR) was performed with SYBR Taq by using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). Briefly, reactions were incubated at 95 °C for 10 s for an initial denaturation, followed by 40 cycles at 95 °C for 15 s and then 60 °C for 1 min. All reactions were run in triplicate, and the relative expression of mRNA or miRNA was calculated using the 2-ΔΔCT method. Human U6 as an endogenous control. The relative gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Cell Counting Kit-8 (CCK-8)
HL-60 and THP-1 cells were seeded onto 6-well plates with six replicate wells and then transfected with TNK2-AS1 shRNA, CELF2 shRNA or sh-NC. After 48 h transfection, HL-60 and THP-1 cells were plated into 96-well plates and incubated for 12 h in a humidified incubator containing 5% CO2 at 37 °C for 24, 48, 72 and 96 h. Then, HL-60 and THP-1 cells were treated with CCK-8 solution (Invitrogen) for 4 h. The absorbance was determined at the wavelength of 450 nm by using microplate reader (Molecular devices, Shanghai, China).
EdU assays
After transfection for 24 h, cell media was replaced with fresh media supplemented with 50 μm EdU reagent for 2 h followed by fixation with 4% phosphate-buffered paraformaldehyde. Then the cells were stained with 100 μL of AdoLo and Hoechst 33 342, respectively. The number of EdU-positive cells was analyzed by fluorescence microscopy (Olympus BX51). The EdU-positive ratio was calculated as the ratio of the number of EdU-positive cells to the total Hoechst 33 342-labeled cells (blue cells).
Flow cytometry analysis
HL-60 and THP-1 cells after transfected were cultured for 48 h, and then digested with trypsin. After centrifugation, cells were harvested and re-suspended with binding buffer. Then, cells were stained with Annexin V-fluorescein isothiocyanate (V-FITC) and Propidium iodide (PI) for 15 min in dark. Fluorescence signals were analyzed directly by flow cytometry using the Cell Quest program (Becton Dickinson, Franklin, NJ).
Nitroblue tetrazolium (NBT) assay
The transfected cells were inoculated in a six-well plate and were collected. A 10 μl aliquot of NBT solution composed of 10 mg/ml NBT (Sigma-Aldrich) and 2 μg/ml PMA (Sigma-Aldrich), was added to each well, and then cells were incubated for 30 min at 37 °C. Then, light microscopy was used for analysis. The positive cells ratio was analyzed by light microscopy
Western blot analysis
Cells were collected and lysed with RIPA lysis buffer (Beyotime, Shanghai, China) for the Western blot analysis. Proteins in the lysates (20 μg per lane) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against EZH2 (1:1000, ab191250, Abcam), CELF2 (1:1000, ab186430, Abcam), ELK1 (1:1000, ab32106, Abcam), CD11b (1:1000, ab13357, Abcam) and CD14 (1:1000, ab182032, Abcam) overnight at room temperature, and then incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody at room temperature for 2 h. The protein bands were visualized using the enhanced chemiluminescence reagents (Millipore, MA, USA). The expression of relative protein was obtained by the gray value ratio of the target protein to the internal reference GAPDH.
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed with the EZ-ChIP Kit (Millipore). Formaldehyde was used to incubate with cells to produce DNA-protein crosslinks. Cross-linked chromatin was sonicated into 200 to 500-base pairs fragments and then the cell lysates were separately incubated with antibodies against ELK1, EZH2 and H3K27me3 (Millipore). The precipitated chromatin DNA were determined by RT-qPCR.
RNA immunoprecipitation (RIP)
RIP assay was performed in HL-60 cells by using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, Billerica, MA, USA). The negative control, normal IgG and antibodies against EZH2 were bond with magnetic beads and cultured with the cell lysates in RIP buffer. The co-precipitated RNAs were detected and quantified by RT-qPCR assay.
Luciferase reporter assay
Cells were plated in 96-well plates and transfected with a compound of 5 ng pRL-CMV Renilla luciferase reporter and 50 ng firefly luciferase reporter. At last, the luciferase activity was measured with the dual luciferase reporter assay system (Promega Corporation, Madison, WI, USA).
Tumor xenografts
All animal care and experiments were carried out in accordance with the guidance of the National Institutes of Health and approved by the Ethics Committee of Zhumadian Central Hospital. We selected 4-week-old NCG mice with severe immunodeficiency for tumor xenotransplantation experiments to study the effect of TNK2-AS1 on tumor proliferation and differentiation. In tumor growth assay in vivo, THP-1 cells stably transfected with TNK2-AS1 sh-RNA (LV-sh-TNK2-AS1) and respective negative control (LV-sh-NC) were subcutaneously injected into the upper back of the nude mice (1 × 107, 200 μL). Tumor length and width were recorded weekly to calculate the tumor volume according to the formula: volume=length × width2/2. After 5 weeks, the tumor was removed for weight measurement, RT-qPCR and Westerrn blot analysis.
Statistical Analysis
All statistical computations were performed by using statistical product and service solutions (SPSS) 23 software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation. Student’s t-test was used to analyze the differential expression. P < 0.05 suggested that the difference was statistically significant.