Patients and specimens
This study collected 128 AML patients who visited PanYu Central Hospital. Meanwhile, 109 healthy volunteers were enrolled into the study as controls. The controls were frequently matched with cases both in age and gender No patients had received any treatment including radiotherapy or chemotherapy before diagnosis. Individuals younger than 18 years old, had other tumors, systemic diseases were excluded from this study. This study was approved by the Ethic committee of PanYu Central Hospital, and written informed consents were also signed by all patients or their family before sampling.
Blood specimens were taken from 128 AML patients and 109 healthy volunteers. Then the blood samples were put into blood collection tube of EDTA and centrifuged to collect leukocytes. Leukocytes were stored at -80℃ until used. Clinicopathological features of the patients were listed in Table 1, including age, gender, WBC, FAB classification, immunophenotype and lymphadenopathy.
Cell culture
Human leukemia cell lines HL-60 and human embryonic kidney cell lines 293T were purchased from the American Type Culture Collection (ATCC; Rockefeller, MD, USA). Cells were cultured in RPMI 1640 media with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin). Then they were incubated at 37℃ humididied atmosphere containing 5% CO2 for cell passage. The media was replaced every two days.
Cell transfection
When the cell density reached approximately 70% confluency, the cells were transfected. KLF6 siRNA, KLF6 siRNA-NC, miR-4262 mimics, miR-4262 mimics NC (negative control), miR-4262 inhibitor, miR-4262 inhibitor NC were purchased from GenePharma (Shanghai, China). They were transfected into HL-60 cells by LipofectamineTM2000 (Invitrogen, CA, USA) according to the manufacturer’s instruction, then incubated at 37℃ humidified atmosphere with 5% CO2 for 6h. Medium was replaced and continuing cultivated for 48h. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify whether the transfection was successful.
qRT-PCR
miRNA from leukocytes was performed with the miRNeasy TM RNA isolation kit (Qiagen, Valencia, CA, USA). RNAs of cells were extracted using Trizol method (TaKaRa, Japan). RNA was reverse-transcribed into cDNA by PrimerScript RT reagent kit (Takara, Dalian, China) according to the instruction. PCR amplification was conducted by SYBR Premix Ex TaqTM II kit in ViiATM 7 real-time fluorescent quantitative PCR system. Expression of KLF6 were normalized to GAPDH, and miR-4262 was normalized to U6. Relative expression of miR-4262 was analyzed by 2-∆∆CT method. Every sample was measured at least three times.
MMT assay
MTT method was used to detect the proliferation of HL-60 cells. Cells transfected with miR-4262 mimics, miR-4262 mimics NC, miR-4262 inhibitor, miR-4262 inhibitor NC were adjusted to 2×104/mL and inoculated on 96-well plates. They were cultured at incubator containing 5% CO2 at 37℃ for 0h, 24h, 48h and 72h respectively. 50μl MTT solution (5mg/mL) were added to cells, then continuously incubated for 4h. Next step, 150μl 20% SDS was added to every well, incubation for overnight at room temperature. Cell proliferation was determined by MTT cell proliferation kit (Cayman Chemical) following the manufacturer’s instruction. MTT enzyme-linked immunometric meter was used to measure OD value (490nm).
Transwell assays
Transwell assays were used for the detection of the migration and invasion abilities of HL-60 cells. Transwell chambers were pre-coated by matrigel (BD Biosciences) and 50μl serum-free medium with BSA were added to the upper compartment, 37˚C dehydration for 2h. Then upper chamber was added 200μl cell suspension and the lower chamber was 500μl DMEM media with 10% FBS. Then cells were incubated at 37˚C incubator with 5% CO2 for 48h. The invasive cells were stained with 0.1% crystal violet for 30 min. Under the microscope the cells were counted in 7 random sights.
Meanwhile, without matrigel transwell assay was conducted to measure the migrative ability of HL-60 cells.
Apoptosis Analysis
Forty-eight hours after transfection, cells were harvested, stained with propidium iodide and anti-annexin-V antibody (Annexin V-FITC Apoptosis Detection kit, BD Biosciences, San Jose, CA, USA) following the manufacturer’s protocol, and stained cells were detected by flow cytometry. The experiments for the apoptosis assay were performed at least three times.
Luciferase reporter assay
In order to verify that KFL-6 is the target gene of miR-4262, KFL-6-3'UTR-WT or KFL-6-3'-UTR-MUT (100 ng) were co-transfected with 100 nM miR-4262 mimics or miR-4262 mimics NC into HL-60 cells using Lipofectamine 2000 (Invitrogen, CA, USA) following the instruction of manufacture. After 48h transfection, HL-60 cells were harvested and measured with a Dual-Luciferase Reporter Assay System Kit (Promega, Madison, WI, USA) according to the manufacturer’s instruction.
Western blot
Transfected cells were lysed using RIPA buffer (Thermo Scientific, Belmont, MA, USA) at 4˚C for 30min. Cells proteins were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Roche) by electroblotting. Nonfat milk was used to block PVDF membrane at 4˚C for overnight. Then it was incubated by primary antibodies at 4℃ for overnight, including anti-KLF6 (1:300), anti-EGFR (1:400) (Abcam, Cambridge, MA, USA), and anti-GAPDH (1:800; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), which was used as the internal reference. Then they were incubated 1.5h at room temperature using second antibody (1:2000, Abcam, China). Target band of protein was showed using ECL Western blotting kit (Millipore, Boston, MA, USA).
Statistical analysis
SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used for data statistics analysis and GraphPad Prism (GraphPad, San Diego, CA, USA) software was applied for plotting. All experiments were repeated at least in triplicate and the data were expressed using mean±SD (standard deviation). Difference between groups was compared by Student’s t-test (Mann-Whitney U test) or χ² test. Two-tailed P<0.05 indicated statistically significant difference.