Clinical samples
Prior to any interventional therapies at the First Affiliated Hospital of Zhengzhou University from March 2018 to April 2019, bone marrow (BM) specimens were harvested during the biopsy from 21 pediatric patients who were newly diagnosed with AML and 10 healthy children as normal controls. The French-American-British (FAB) classification was used as the standard criteria for the diagnosis of AML patients [19, 20]. Signed consent for clinical studies was signed by their guardians prior to the study and our study was approved by Institutional Ethics Board of the First Affiliated Hospital of Zhengzhou University.
Cell culture and transfection
The AML cell lines (HL60 and K562), as well as human bone marrow stromal cell line (HS-5 ) were get from the American Type Culture Collection (ATCC) (Manassas, VA, USA). These cells were cultivated in RPMI-1640 medium (BOSTER, Wuhan, China) complemented with 10% heat-inactivated fetal calf serum (ExCell Bio, Shanghai, China), together with 1% penicillin/streptomycin in a water-saturated culture incubator flushed with 5% CO2 and 95% air under 37°C. ADR-resistant HL60 (HL60/ADR) and K562 (K562/ADR) cells were provided by Institute of Haematology, Chinese Academy of Medical Sciences (Tianjin, China). To keep the drug resistance phenotype, HL60/ADR and K562/ADR cells were incubated in culture medium containing 0.1 µM ADR (Sigma, St Louis, MO, USA) and further cultivated without ADR for 2 weeks before experiments.
pcDNA-TUG1 (TUG1), control vector (Vector), siRNA specially targeting TUG1 (si-TUG1), control siRNA (si-NC), miR-186-5p mimics (miR-186-5p), NC-mimic (miR-NC), miR-186-5p antigomir (anti-miR-186-5p), and antigomir control (anti-miR-NC) were all bought from GenePharma Co., Ltd. (Shanghai, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was then taken to perform transient transfection.
Cell viability assay
The cell counting kit-8 (CCK-8) assay was used for cell viability evaluation. 96-well plates were used to carry the logarithmically growing HL60/ ADR and HL60 cells with a density of 5 × 103 cells per well and all the cells were exposed to 8 μM ADR or infected with si-TUG1, TUG1, miR-186-5p, anti-miR-186-5p, or matched controls in the absence or presence of 8 μM ADR. After incubated for the indicated time, the cells were fostered for another 3 h after adding 10 μL CCK-8 solutions. A MultiSkan3 ELISA Reader (Thermo Fisher Scientific, Waltham, MA, USA) was adopted to record the cell viability.
Quantitative real-time polymerase chain reaction (qRT-PCR)
RNAiso Plus (Takara, Dalian, China) was taken to extract total RNA. Using SuperScript III reverse transcriptase (Invitrogen), the synthesis of first strand cDNA was carried out. To examine TUG1 and EVI-1 mRNA expressions, qRT-PCR was implemented on a Chromo4 instrument (Bio-Rad) with THUNDERBIRD SYBR qPCR mix (Toyobo, Osaka, Japan), with GAPDH as the normalization. Additionally, miR-186-5p expression was examined using miScript SYBR Green PCR Kit (Qiagen) on a Chromo4 instrument (Bio-Rad) and normalized to U6 small nuclear RNA (snRNA). The fold changes were detected according to the 2-ΔΔCt method.
Western blot analysis
RIPA lysis buffer (KeyGEN, Nanjing, China) containing phenylmethylsulphonyi fluoride (PMSF) was used to lyse collected samples and the protein content was quantified by using the BCA™ Protein Assay Kit (Beyotime, Shanghai, China) . The 10% SDS-JPAGE gel was used to separate collected cell lysates, and separated brands were then electro-transferred onto nitrocellulose membranes. Next, the membranes were exposed to 5% skimmed milk for 1 hour. Then, the blocked membranes were probed with primary antibodies for 12 hours at 4°C and secondary antibody marked by horseradish peroxidase (Abcam, Cambridge, MA, USA) at room temperature. An ECL detection reagent (Solarbio, Shanghai, China) was implemented to measure the signals. The primary antibodies in this study include proliferating cell nuclear antigen (PCNA), Bcl-2, EVI-1 (Abcam); P-glycoprotein (P-gp), PI3K, phosphorylated PI3K (p-PI3K), phosphorylated mTOR (p-mTOR), phosphorylated Akt (p-Akt) and β-actin (Abcam).
Luciferase reporter assay
The fragments from TUG1 cDNA encompassing two potential miR-186-5p-targeting sites and its two mutated (MUT) counterparts were synthesized and subcloned into p-MIR-reporter plasmid (Thermo Fisher Scientific) to produce TUG1 wild type, TUG1 mutant type 1 and TUG1 mutant type 2. Subsequently, HL60/ADR and HL60 cells were cotransfected with TUG1 wild type, TUG1 mutant type 1, or TUG1 mutant type 2 and anti-miR-186-5p, miR-186-5p, or respective controls by means of Lipofectamine 2000 (Invitrogen). A luminometer was used to measure the luciferase activity under the Luciferase Reporter System (Promega, Madison, WI, USA).
Statistics
GraphPad software 6.0 (GraphPad Inc., San Diego, CA, USA) was taken for statistical analysis. All results were displayed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and two-tailed Student’s t-test and were conducted to determine the significance of differences. P values < 0.05 between differences were regarded statistically significant.