Materials
RIPA lysis and extraction buffer was purchased from Beyotime Biotechnology Co., Ltd. (Jiangsu, China). PhosSTOP Phosphatase Inhibitor Cocktail Tablets were purchased from Roche (Shanghai, China). Nuclear protein extraction kit was purchased from Active Motif Co., Ltd. (Shanghai, China). Protease inhibitor cocktail set III was purchased from EMD Biosciences (Germany). FAM168A antibody [FAM168A (E-13)] was purchased from Santa Cruz (U.S.A.). AKT1 antibody, p-AKT1 antibody, β-actin antibody, HRP-conjugated secondary antibodies, goat anti-rabbit IgG and horse anti-mouse IgG were purchased from Cell Signaling (U.S.A.). Fetal bovine serum, IMEM media, and trypsin were purchased from Invitrogen (U.S.A.). BCA protein assay kit was purchased from Pierce Corporation (U.S.A.). SDS, Tris-Cl, EDTA, and acrylamide were purchased from Sigma (U.S.A.).
Human chronic myeloid leukemia cell line K562 was purchased from China Center for Type Culture Collection. K562 cells were cultured in IMEM media containing 10% fetal bovine serum at 37 °C with 5% CO2. Cells in logarithmic growth phase were selected.
Twenty-one male NOD/SCID mice, 5–6 weeks of age, weighing 25–30 g, were purchased from Nanjing University-Nanjing Institute of Biomedical Research (Qualification No.: 32002100001171), and were housed in specific-pathogen-free (SPF) laminar air flow rooms [equipment qualification certificate: SYXK (Guangdong) 2012–0119] at Shenzhen Institute of Advanced Technology. Animals had free access to food and water. The study was approved by the Laboratory Animal Ethics Committee of the Shenzhen Advanced Institute of the Chinese Academy of Sciences.
Three patients, 2 males (6–10 years old) and 1 female (6–10 years old), with CML hospitalized in the Department of Hematology and Oncology at our hospital from March 2018 to May 2018 were included. These patients were newly diagnosed with CML. The clinical features of patients are shown in the Additional file
1: Table S1. Peripheral blood (2 mL) were collected from these patients before treatment. During the same time period, 3 healthy children underwent annual physical examination were selected, including 2 males and 1 female, 6–9 years of age, and 2 mL of peripheral blood were collected. The study was approved by the hospital’s ethics committee. Informed consents were signed by the parents of every child before the blood collection.
Methods
Specimens collection
Inclusion criteria for CML patients were as follows: patients who had typical elevated white blood cell counts and abnormalities in classification, splenomegaly with Ph chromosomes or their variant karyotypes, and BCR-ABL1 fusion gene positivity according to the clinical diagnosis. Inclusion criteria for healthy controls were as follows: healthy children who had no underlying diseases. 2 mL of peripheral blood was collected from the patients and the healthy controls using EDTAK2 anticoagulant tubes and stored at 4 °C until use.
Isolation of peripheral blood mononuclear cells
The whole blood was diluted with equal amount of D-Hank’s solution or PBS. 2 mL of the diluted blood was added along the wall of the tube into 1 mL of lymphocyte isolation solution and centrifuged at 400 x g for 20 min at 15 °C. The white lymphocyte layer was collected using a capillary pipette, placed in another centrifuge tube and washed with PBS for 3 times to collect the cells.
Cell culture
K562 cells were cultured in IMEM media (Thermo Fisher Scientific, U.S.A.) containing 10% fetal bovine serum (Thermo Fisher Scientific, U.S.A.), and incubated at 37 °C with 5% CO2. The cells in logarithmic growth phase were selected.
FAM168A siRNA interference
The siRNA sequence for the FAM168A exon (5′- GCCUCAUGUCAUCCACCAU − 3′) and the negative control siRNA sequence were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. (Guandong, China). K562 cells were seeded into 6-well plates at a density of 3 × 105 per well. Next day, the siRNAs and Lipofectamine 2000 were dissolved in serum-free media, mixed, and incubated at room temperature for 20 min. siRNAs/Lipofectamine 2000 mixtures were added to the cells, and cultured for 48 h.
RNA isolation and first strand cDNA synthesis
5 × 105 PBMCs and K562 cells were collected and homogenized in 1 mL Trizol. 0.2 mL chloroform was added, vortexed, and centrifuged at 2,000 x g for 15 min at 4 °C. The upper aqueous phase was collected and 0.5 mL isopropanol was added. After high-speed centrifugation, the supernatant was discarded. The RNA pallet was washed with 75% ethanol and centrifuged to remove the supernatant. The pellet was dried and dissolved in DEPC-treated water. RNA concentration and quality were measured, and stored at − 70 °C. For reverse transcription, 2 U DNase I was added to 1 μg RNA and incubate at 37 °C for 30 min to remove the genomic DNA. EDTA was added to stop the reaction. Oligo (dT) primers and reverse transcriptase were added to the RNA and incubate at 42 °C for 60 min. The reaction was stopped by heating at 70 °C for 5 min.
Real-time quantitative PCR
A total of 20 μL of reaction system was used and 3 replicates were set for each sample. Real-time PCR program was as follows: 95 °C for 10 min; 95 °C for 15 s, 60 °C for 1 min, 40 cycles. The melting curve was plotted at 70 °C to 95 °C. The experiment was repeated three times. β-Actin was used as an internal reference control. The gene expression was calculated by comparative Ct method (2-ΔΔCt method). The ΔΔCt was calculated as: ΔΔCt = [(target gene Ct - internal reference Ct) treatment group - (target gene Ct - internal reference Ct) control group]. Real-time PCR detection primers for FAM168A were: 5′-GAACTCGTCTTC CTGTGGCA-3′ (FAM168A-F) and 5′-GGGGTGGAGCAGTGTTACTC-3′ (FAM168A-R). Detection primers for β-Actin were: 5′-CTCACCATGGATGATGATATCGC-3′ (β-Actin-F) and 5′-AGGAATCCTTCTGACCCATGC-3′ (β-Actin-R).
K562 cells were cultured in 10-cm cell culture dishes. When the cells reached 80% confluence, the media were removed, and the cells were washed with 5 mL of ice-cold PBS containing phosphatase inhibitor. After centrifugation, the supernatant was removed. Nuclear protein extraction kit was used to extract nuclear protein. In brief, the cells were gently resuspended in 500 μL 1× hypotonic buffer, transferred to a 1.5 mL ice-cold centrifuge tube, and incubated on ice for 15 min. 25 μL detergent solution was added, vortexed for 10 s, and centrifuged at 14,000 x g for 30 s at 4 °C. The pellet was resuspended in 50 μL complete lysis buffer, vortexed for 10 s, incubated on ice for 30 min, vortexed for 30 s, and centrifuged at 14,000 x g for 10 min at 4 °C. The supernatant was used for Western blot analysis.
Western blot
6 × SDS loading buffer [0.35 M Tris-HCl, pH 6.8, 36% glycerol, 0.012% bromophenol blue, 10.28% SDS (w/v), 0.6 M DTT] was added to10 μg protein, boiled at 100 °C for 5 min, and cooled on ice. The proteins were loaded onto a 10% SDS-PAGE, and separated by electrophoresis. The proteins were transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST overnight at 4 °C, and incubated with primary antibodies for 2 h. The membranes were washed three times (5 min each) with TBST, and then incubated with the corresponding secondary antibodies for 1 h. The membranes were washed three times with TBST, and then rinsed three times with ddH2O. The membranes were reacted with ECL chemiluminescence detection reagents in dark, and the X-ray films were developed and imaged.
The protein sequence of human FAM168A was downloaded from the GenBank (number: NP_055974.1) from the National Center for Biotechnology Information (NCBI) database, and the motif analysis of FAM168A was performed using Scansite database (
http://scansite.mit.edu/) [
12].
Immunoprecipitation
K562 cells were cultured in a 10 cm dish. After the cells reached 100% confluence, the media were removed and the cells were washed once with PBS. 400–500 μL of RIPA lysis buffer (50 mM Tris-Cl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1% sodium deoxycholate; 0.1% SDS; 1 mM sodium orthovanadate; 10 mM sodium fluoride; 1% protease inhibitor cocktail) was added to the cells and incubated on ice for 10 min. After sonication, the lysates were centrifuged at 13000 x g for 10 min at 4 °C, and the supernatants were collected. A small amount of supernatants were used as an “Input sample” for Western blot analysis. The remaining supernatants were divided into four equal portions. The first three portions were incubated with 1–2 μg of FAM168A, c-ABL1, or AKT1 antibody overnight at 4 °C, respectively. The fourth portion was incubated with the same amount of rabbit IgG as a negative control. The next day, 20 μL of protein G beads were added to each tube and incubated at room temperature for 30 min. The magnetic beads were washed three times with RIPA lysis buffer. 40 μL of 2 × SDS loading buffer was added to the beads, heated at 95–100 °C for 5 min, and centrifuged at 13000 x g for 1 min to collect the supernatant. The supernatant and the Input sample were analyzed by Western blot.
Flow cytometry
K562 cells were seeded into 6-well plates, transfected with FAM168A siRNA or control siRNA, and cultured for 48 h. Cells were collected and washed with PBS. Cells were resuspended in 70% ice-cold ethanol, fixed for 2 h, and centrifuged to remove supernatants. The cells were washed twice with 0.5% BSA and 0.1% Triton X-100 in PBS. Propidium iodide was added and incubated for 30 min, and the cell cycle DNA content was measured by flow cytometry (Beckman Coulter).
CML mouse model
Twenty-one male NOD/SCID mice, 5–6 weeks old, weighing 25–30 g, were housed in a SPF laminar flow chamber. The mice were randomized into 3 groups of 7: K562/Control group, K562/siFAM168A group and negative control group. The mice in K562/siFAM168A and K562/Control groups were injected with K562 cells transfected with FAM168A siRNA or control siRNA, respectively, via the tail vein. The cell concentration was 5 × 106 / mouse, and the injection volume was 0.2 mL. The mice in the negative control group received saline injection (0.2 mL/mouse) via the tail vein. The mice in negative control group were sacrificed by neck dislocation at the end of experiment.
Survival analysis
The color of the fur, body weight, tumor growth, and survival duration of NOD/SCID mice in each group were recorded, and the tumor formation rate and survival rate were compared.
Statistical analysis
The experimental data were analyzed by SPSS19.0 statistical software, and the data were expressed as mean ± SD (standard deviation). Student t-test was used for two-group comparison and p < 0.05 was considered statistically significant.