2.1 Tissue samples and ethics statement
Bone marrow samples were obtained from patients with newly diagnosed AML (n = 39) and healthy subjects (n = 39), from Department of Hematology, the First Affiliated Hospital of USTC. The monocytes were available from these samples through standard Ficoll-Hypaque density gradient centrifugation. This research was conducted with informed consent from all participators and endorsed by the Ethics Committee of the First Affiliated Hospital of USTC, University of Science and Technology of China.
2.2 Cell culture
The China Center for Type Culture Collection (CCTCC, Wuhan, China) was the provider of human AML cell lines (KG1, NB4, NOMO-1, and HL60) and bone marrow hematopoietic stem cell line (CD34). Aforementioned cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA), 100 U/ml penicillin, 100 µg/ml streptomycin (Hyclone, Logan, UT, USA) and L-glutamine (2 mM; Gibco, Carlsbad, CA, USA) in 5% CO2 at 37 °C.
2.3 Treatment with RNase R and qRT-PCR
Firstly, total RNA extracted from tumor tissues and cells by Trizol reagent (Takara Biotechnology Co., Ltd., Dalian, China) was incubated with or without 3 U/mg RNAse R (Epicentre Technologies, Madison, WI, USA) for 20 min at 37 °C. Secondly, first-strand cDNA was generated adopting the Prime-Script RT reagent kit (TaKaRa, Dalian, China). Thirdly, qRT-PCR was operated on the StepOne Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA) by the SYBR Green PCR kit (Takara Bio, Inc., Otsu, Japan). In this context, GAPDH or U6 was employed as endogenous controls. The primers’ sequences were shown in Table 1.
Table 1
Sequences used for qRT-PCR.
circ_0000005
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F: 5’-AGGGGGAGCTGAGAGATCA-3’
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R: 5’-TCTTTCCGAGACATTTGCTG-3’
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miR-139-5p
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F: 5’- GCCTCTACAGTGCACGTGTCTC-3’
R: 5’- CGCTGTTCTCATCTGTCTCGC-3’
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Tspan3
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F: 5’-CATGTGATCTGGGCCG-3’
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R: 5’-CGCCAGTGATGAGGAG-3'
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U6
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F: 5’-CTCGCTTCGGCAGCACA-3’
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R: 5’-AACGCTTCACgAATTTGCGT-3’
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GAPDH
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F: 5’-ACACCCACTCCTCCACCTTT-3’
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R: 5’-TTACTCCTTGGAGGCCATGT-3’
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Abbreviation: F stands for forward; R stands for reverse.
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2.4 Subcellular fractionation
Cells were lysed in the RIPA lysis (Solarbio, Beijing, China) and incubated for on ice 10 min. The cell lysate was separated by high-speed centrifugation (3 min, 12000 g). Cytoplasmic RNA was obtained in the supernatant and nuclear RNA in the nuclear pellet. Purified RNAs were estimated by qRT-PCR. Specifically, GAPDH and U6 worked as cytoplasmic and nuclear endogenous references, respectively.
2.5 Cell transfection
pcDNA3.1-circ_0000005, pcDNA3.1 empty vector, siRNA specific to circ_0000005, control siRNA (si-NC), miR-139-5p mimics and inhibitors (miR-139-5p and miR-139-5p in), mimics control and inhibitors control (miR-NC and NC inhibitors) were synthesized by Ribobio (Guangzhou, China). AML cells were transfected with the vectors or oligonucleotides mentioned above using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) in compliance with the manufacturer's instruction.
2.6 Cell counting kit-8 (CCK-8 assay)
After transfectoin, AML cells were inoculated onto 96 well plates (2000 cells in 100 µL serum medium / well ). AT different time points (1, 2, 3 and 4 h after inoculation), 10 µL CCK-8 solution (Dojindo, Tokyo, Japan) was dripped into each well. After the cells were incubated for 1 h at room temperature, the absorbance of each well at 450 nm was assessed by a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
2.7 Flow cytometry analysis
Cells rinsed with pre-cooled phosphate buffer saline (PBS) were resuspended in 500 µL binding buffer, and then stained with 5 µL fluorescein isothiocyanate (FITC)-Annexin-V (Sigma-Aldrich, Louis, MO, USA) staining solution and 10 µL propidium iodide (PI) staining solution (50 µg/mL, BD Biosciences, San Diego, CA, USA) for 15 min, away from light. Next, the cell apoptosis was estimated by the a flow cytometer (FACScan®; BD Biosciences, San Diego, CA, USA). The data were processed with FlowJo software (TreeStar, San Carlos, CA, USA).
2.8 Migration and invasion assays
Transwell chambers (pore size, 8 µm; Corning, NY, USA) coated with or without Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were used for invasion and migration assays, respectively. In brief, cell suspension in FBS-free RPMI-1640 medium (2 × 104 cells / well) and RPMI-1640 medium containing with 20% FBS (600 µL / well) were added into the upper and lower chambers, respectively. The cells were cultured for 48 h in 5% CO2 at 37 ℃, and thereafter, migrated and invaded cells were recorded.
2.9 Western blot assay
RIPA lysis (Solarbio, Beijing, China) was adopted to isolate the total proteins from cells. The protein concentrations were quantified with BCA Protein Assay Kit (Beyotime, Haimen, China). The protein samples were then mixed with loading buffer, and heated in boiling water for 5 min for denaturation. After that 20 µg protein in each group was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% skim milk, and then incubated with diluted primary antibodies including anti-Tspan3 (ab151299, 1:1000, Abcam, China) and anti-GAPDH (ab8245, 1: 2000, Abcam, China) for 12 h at 4 ℃. After that, the membrane was incubated with HRP-conjugated secondary antibody (ab205718, 1:2000, Abcam) at room temperature at room temperature for 2 h. Finally, the protein bands were visualized by Enhanced Chemiluminescence Western Blotting Substrate (Dongguan Biotech, Shandong, China).
2.10 Luciferase reporter gene assay
Wild type (WT) and mutant (MUT) fragments of circ_0000005 sequences were inserted into pMIR-REPORT vectors (Promega, Madison, WI, USA). Similarly, WT and MUT fragments of Tspan3 3’-UTR with the binding sites for miR-139-5p were cloned into pMIR-REPORT vectors. After the reporter vectors were constructed, the cells were co-transfected with these reporter vectors and miR-139-5p mimics or miR-NC. 48 h later, dual-luciferase reporter gene assay system (Promega, Madison, WI, USA) was employed to detect the luciferase activity in the cell lysate of each group.
2.11 RNA pull-down assay
Purified RNAs were labeled employing biotin with Pierce™ RNA 3′End Desthiobiotinylation Kit (Life Technologies, Baltimore, MD, USA). Cells transfected with biotin-labeled miR-139-5p (Bio-miR-139-5p) (GenePharma, Shanghai, China) or biotinylated RNAs (NC-Bio) (20 nM) by Lipofectamine™ 3000 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) were harvested. The cells were added with lysis buffer, and then incubated on ice for 5 min. After that, the lysis was centrifuged and the cell debris was discarded. After that, magnetic beads were was added and incubated at 4 °C for 2 h and then RNA was purified by RNeasy Mini Kit (QIAGEN, Duesseldorf, Germany). Ultimately, qRT-PCR was performed to detect the expression of circ_0000005.
2.12 RNA immunoprecipitation (RIP) assay
The Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was applied to RIP assay. In short, RIPA lysis buffer (Beyotime, Shanghai, China) was employed to lyse T-ALL cells, and 200 µL cell lysates were incubated with 5 µg antibody against anti-Argonaute 2 (Ago 2) (Abcam, Cambridge, UK) or anti-immunoglobulin G (IgG) (Abcam, Cambridge, UK) and rotated at 4 °C overnight. After being treated with proteinase K (Beyotime, Shanghai, China), immunoprecipitated RNA was extracted by the RNeasy MinElute Cleanup Kit (Qiagen, Shanghai, China) and reversely transcribed adopting Prime-Script RT Master Mix (TaKaRa, Dalian, China). Eventually, qRT-PCR was utilized to detect the abundance of circ_000005 and miR-139-5p.
2.13 Statistical analysis
Data were analyzed on the basis of IBM SPSS 17.0 software (IBM, Armonk, NY, USA) and GraphPad Prism 7.0 (GraphPad Software, Inc. La Jolla, CA, USA), and showed in the form of mean ± SD. The difference between groups was observed by Student’s t-test, χ2 test or Wilcoxon test. The correlations among circ_0000005, miR-139-5p and Tspan3 expressions in AML samples was evaluated through Pearson's correlation analysis.