Ethical statement
All subjects in this study signed written informed consent which was protocolled according to the guidelines. All experiments of this study were supported by the Clinical Research Ethics Committee of Wenzhou Central Hospital.
Clinical sample
The samples were collected from 60 patients who were diagnosed as AML (including 30 FLT3-ITD+ AML patients, 30 FLT3-ITD- AML patients) and 30 healthy donors. Among those participants, the gender ratio approached 1:1 and the ages of them were between 30 and 60. All materials were admitted to Wenzhou Central Hospital. The samples were exacted out of patients and volunteers by operating and then were stored at −80°C for use.
Cell culture and transfection
Human cell line Molm-13 (FLT3-ITD+ cell line), MV4-11 (FLT3-ITD+ cell line), THP-1 (FLT-ITD wild type) and U937 (FLT-ITD wild type) were obtained from ATCC (Manassas, VA, USA) and was cultured in DMEM. Medium supplemented were with 10% FBS (fetal bovine serum) storing at 37°C with 5% CO2.
The cDNA of PSMA3-AS1 was synthesized according to the sequence obtained from lncBase database, and then cloned into pcDNA3.1 plasmid using TA Cloning™ Kit (Thermo Fisher Scientific). PcDNA3.1 plasmid vectors, pcDNA3.1-PSMA3-AS1 (oe-PSMA3-AS1), siRNA- PSMA3-AS1 (si-PSMA3-AS1), pcDNA3.1-ATG16L1 was obtained from Genechem (Shanghai, China). MiR-20a-5p mimics and miRNA negative control were synthesized by RiBoBio (Guangzhou, China). Transfection of blank plasmid was applied to the control group (Ctrls). Cells were transiently transfected by Lipofectamine 2000 reagent (Invitrogen) in the basis of the manufacturer’s protocol. After being transfected for 24 h, cells were obtained and used for experiments.
Expression profile analysis of lncRNAs
The lncRNA chip analysis data came from the GEO database (GSE103828). The 10 samples got analyzed using lncRNAs chips. In the study presented here, 5 pairs of AML patients and iron deficiency anemia (IDA) controls were screening by microarray. Labeled RNAs were scanned and acquired by employing an Agilent-079487 Arraystar Human LncRNA microarray V4 (Agilent Technologies, USA), and array images were analyzed by Agilent Feature Extraction software (version 11.0.1.1, USA). The differentially expressed lncRNAs were selected according to the fold-change cut-off (fold change≥2) and P-value <0.05.
The Cancer Genome Atlas (TCGA) database analysis
RNAseq data (level 3) of 151 AML samples (38 treated as controls and 113 untreated as study) were obtained from The Cancer Genome Atlas (TCGA) dataset (https://portal.gdc.com) and corresponding clinical information. m6A-related genes were derived from Juan Xu et al.'s study on the molecular characterization and clinical significance of m6A regulators across 33 cancer types.
The above results were achieved with the R (v4.0.3) packages ggplot2 and pheatmap.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA extraction from purified cells lines was performed by using TRIzol (Life Technologies, Gaithersburg, MD, USA). 1 μg of RNA was treated with DNase I prior and then reverse transcription into cDNA. qRT-PCR assays functioned by using Platinum® SYBR® Green qPCR SuperMix (Life Technologies) which made housekeeping gene GAPDH or U6 the internal control. The 2-ΔΔCt (comparative threshold cycle) method was employed to identify the relative quantification of gene expression levels. RiboBio (Guangzhou, China) was employed to synthesize the premier sequences and the results were shown in Table S1.
Western blot
Cell lysates were disposed by a detergent buffer. Protein concentrations were measured with the BCA Protein Assay on the basis of the manufacturer’s manual (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (80 μg) of protein were isolated by 12% SDS-PAGE gels electrophoresis and were transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA). After being blocked by 5% nonfat dry milk (NFDM), membranes achieved incubated overnight at 4 °C with a 1:1000 dilution of anti-ATG16L1 (ab187671, Abcam, Cambridge, MA, USA) or GAPDH (ab8245, Abcam). After additional incubation with a 1:1000 dilution of a goat anti-mouse horseradish peroxidase-linked antibody (ab6785) for 1 h, protein bands on the blots were measured by the use of Image J software (Bio-Rad Laboratories, USA).
MTT assay
The cell proliferation of each treatment group was determined by measuring the amount of MTT reduced to formaldehyde. MTT reagent was then supplemented, followed by cell incubation for 3 h. The solution was decanted, and 100μL DMSO was added for dissolution of purple formazan crystals. The absorbance of the resulting solution was measured at 570nm with a microplate (Bio, USA).
Edu Staining
To measure proliferation potential of cells, cells were subjected to the Cell-Light TM EdU imaging detecting kit (RiboBio, Shanghai, China). Briefly, each well of a 96-well plate contained cells (1×105/well). After being cultured with 10 mM EdU for 2 h, all the Cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 and stained by Apollo solution for 30 min in the dark. Subsequently, cell nuclei got stained by DAPI solution.
Cell apoptosis analysis
Cells were harvested from different groups for apoptosis analysis after double staining of the cells with Annexin-V and Propidiumiodide (PI) via using Annexin-V-FLOUS staining kit in the basis of the manufacturer's protocol (Roche, Mannheim, Germany).
Dual luciferase activity assay
Subcloned into luciferase reporter psiCHECK2 (Promega, Madison, WI). On 96-well plates, cells were seeded in the density of 3×104 cells per well for 24 h in triplicate before transfection and then transfected with wild-type or mutated reporter vectors, and miRNA mimics or negative control. Lysates were harvested after being transfected for 24 h.
RNA Stability Assays
MV4-11 cells were treated with actinomycin D at a final concentration of 5 μg/ml, and the experiment was divided into control group and METTL3 knockout group. After 0, 2 and 4 hours of treatment, cells in each group were collected for RNA isolation. Then, the expression of PSM3-AS1 in each treatment group was detected by qRT-PCR.
RIP assay
RIP was employed by using a Magna RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) on the basis of the manufacturer's instructions. MV4-11 cell lysates containing PSMA3-AS1, ATG16L1 and miR-20a-5p got prepared and incubated with anti-argonaute2 (Ago2) antibody (Millipore). NC was set with Normal mouse IgG (Millipore).
Animals
6-week-old nude mice were obtained from Shanghai Lab. Animal Research Center (Shanghai, China). A total of eighteen mice were randomly divided into three groups, and were injected with serum free cell suspensions of MV4-11 cells that transfected with vector (as negative control, named Ctrls) or si-METTL3, respectively. Tumor size was observed every 6 days for 42 days and was calculated by the following formula: 1/2×L2×W (L, length (mm), W, width (mm) of the tumor). When the experiment approached termination (the 42st day), the tumor in body of each mouse was excised and was weighted.
Immunohistochemistry (IHC)
Longitudinal sections of tumor specimens were fixed in 10% formalin, embedded in paraffin, and then completed by deparaffinizing with xylene and hydrating with an ethanol gradient. After being successively incubated with antigen retrieval solution (Shunbai, China) and 3% H2O2 for 30 min, the slides were washed with water and cultured with the primary antibody anti-ki67 (1:100) and anti-TUNEL (Sevier, China) overnight at 4°C. Nonimmunized serum was utilized to dispose negative controls with discarding primary antibody. The next day, secondary antibody (Beijing Biosynthesis Biotechnology Co. Ltd., Beijing, China) was used to rinse and incubate slides, which then were followed by 3, 3′-diaminobenzidine (DAB) and hematoxylin staining, respectively. Image-J software was performed to analyze the results of DAB staining.
Statistical analysis
All the data were analyzed by using GraphPad Prism 6.0 and they were presented as the mean ± SD (standard deviation). The in vitro experiments were performed three times. Difference between two groups were identified by Students’ T-test and to measure differences among three groups or more, one-way ANOVA was employed. P-values less than 0.05 was considered as a statistically significant difference.