Cell lines and culture
K562 cell were purchased from Procell Life Science& Technology Co. Ltd. All cells are cultured in RPMI 1640 media containing 10% fetal calf serum and 1% antibiotic (penicillin and streptomycin) at 37 °C in a 5% CO2 incubator.
Transfection
K562 cells were transfected with lipofectamine2000 (Invitrogen). Transduce according to manufacturer's instructions and incubate leukemia cells in Opti-MEM. K562 cells/well (2*105) with hsa-miR-22 miRNA mimic and negative mimic control were used to increase miRNA activity. miR-22 inhibitor were used to inhibit miRNA activity. The cells were harvested 48 hours after transfection. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of miR-22.
RNA extraction, transcription (RT) and qRT-PCR
Total RNA from culture cells was extracted using the Trizal reagent (Invitrogen) following the manufacturer’s instruction. cDNA was synthesized using TAKARA Reverse KIT. SYBR Premix Ex Taq. II kit (TAKARA, Japan) was used for qPCR. An Applied Biosystems (Foster City, CA, USA) 7500 Fast Real-Time PCR system was utilized for qRT-PCR analyses. NCBI/Primer BLAST designed human p53, TET2 and GADPH cDNAs sequence-specific primers as ESI table S1. The relative expression of the target gene (2−ΔΔCt) is normalized for GADPH.
Western blotting
K562 cells from each group were collected and washed three times with PBS, and then the supernatant was discarded. Afterwards, 100 μL lysate was added in cells, followed by incubation on ice for 5 min and centrifugation at 10000 g for 10 min. Then, Collect supernatant and determine total protein concentration by Coomassie brilliant blue (Shanghai Majorbio Co., Ltd. Shanghai, China). Cell lysates containing 50 lbs of protein were separated in 10% Tris-HCl gel and transferred to blister membranes, and then protein blocking was performed in IX Tris-buffer saline (TBS) with 5% skim milk. Subsequently, the blot was incubated with the primary antibody (Proteintech; 1:1,000) dilution overnight. The membrane was washed in TBS-T and then combined with horseradish peroxidase (Proteintech; 1:5,000 in TBS). Finally, the film was cleaned with TBS-T and developed using ECL substrate. X-ray films observed protein bands. The protein measurement standard is GADPH protein
Cell cycle and apoptosis assay
Cells were collected after miR-22 mimics and miRNA inhibitor infection for 48 h and then re-suspended in binding buffer. Apoptotic cells were detected by Annexin V/PI double staining flow cytometry (FCM) kit (KeyGEN BioTECH Jiangsu China) and a ten-color flow cytometer (Beckman Coulter Gallios, Inc. USA).
Cell proliferation assay
The proliferation was tested by using CCK-8 assay (Dojindo Laboratories, Japan). First, Cells were collected after miR-22 mimics and miRNA inhibitor infection for 48 h and then re-suspended in RPMI-1640 medium of 100 μL, cells were added with 100μL of CCK-8 solution. Subsequently, the plates were inch bated for 2 h and incubate at 37°C for 4 hours in the dark. Determination of absorbance with 450 nm spectrophotometer (Bio-Tek, Epoch. Inc, USA).
Dual-luciferase assay
The TP53 gene was cloned into the vector pcDNA3.1 (+) to construct the vector TP53- pcDNA3.1 (+) (Fenghbio, Changsha, China). The WT vector or the empty vector and miR-22 mimics or NC was contradicted into 293T cells using Lipofectamine 3000 reagents. Forty-eight hours after transfection, the luciferase activity was assessed using a Dual-luciferase Reporter Assay System (Lux-T020, BLT).
Statistical analysis
Data are expressed as mean ± standard deviation (SD), and the calcium content of each experimental group. Differences between groups were analyzed by single or double variance, and then Prism statistical analysis software (Graph Pad software) was used to conduct Bonferroni multiple comparison test. P <0.05 is considered a significant difference.