Cell culture
The Ana-1 murine macrophages were purchased from American Type Culture Collection (ATCC). Ana-1 murine macrophages were incubated in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin and streptomycin in 95% humidified atmosphere and 5% CO2 at 37˚C.
For the establishment of an in vitro pneumonia model, the Ana-1 murine macrophages were induced by 1 µg/ml LPS (10; 31), and they were randomly separated into 4 groups, including control group, LPS group, LPS+antagomiR-103a-3p group and LPS+antagomiR-103a-3p+siRNA PTEN group.
Serum samples
The blood samples were obtained from 29 healthy children (16 males and 13 females, aged from 3 to 13 years old) and 29 patients with pneumonia (19 males and 10 females, aged from 3 to 13 years old) in Guizhou Provincial People’s Hospital (Guizhou, China) between Mar, 2016 and Feb, 2018. Serum samples were obtained via centrifugation at the speed of 1,000 x g for 10 min, then the supernatant was collected for the subsequent experiments. The present study was approved by the Ethical Committee of Guizhou Provincial People’s Hospital (approval no. 2016011605). All the parents or the legal guardians of the participants provided written informed consent prior to the conduction of the present study.
Transient transfection
antagomiR-103a-3p, antagomiR-negative control (NC), siRNA, siRNA1 PTEN and siRNA2 PTEN were synthesized by GenePharma Corporation (Shanghai, China). Cell transfection of antagomiR-103a-3p, antagomiR-NC and/or siRNA, siRNA PTEN was conducted using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s protocols.
Cell viability assay
Cell viability was determined by a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) in accordance with the manufacturer's protocols. Ana-1 murine macrophages (5x103 cells/well) were placed in 96-well plates and incubated for 24 h at 37˚C. After that, 10 µl CCK-8 solution was added to each well of the 96-well plates. Ana-1 murine macrophages were incubated for 2 h at 37˚C. The absorbance at 450 nm was recorded.
Cell apoptosis assay
Cell apoptosis was detected by Annexin-V/Dead Cell Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were then diluted in 100 µl 1X Annexin-binding buffer to 1x106 cells/ml. Annexin V (5 µl) and propidium iodide (1 µl) were added to the above cell suspension. Cells were placed at room temperature for 15 min. Afterwards, annexin-binding buffer (400 µl) was added. Stained cells were analyzed by BD FACSCalibur flow cytometer (BD Biosciences).
ELISA
The Ana-1 murine macrophages (5x105 cells/well) in 4 groups were plated in their corresponding 24-well plate, followed by incubation in 95% humidified atmosphere and 5% CO2 at 37˚C overnight. Afterwards, the protein levels of TNF-α, IL-6 and IL-1β from the supernatants of Ana-1 murine macrophages were assessed by ELISA kits for mouse TNF-α (BMS607-3) which was purchased from Invitrogen (Carlsbad, CA, USA), and ELISA kits for mouse IL-6 (RAB0308) and IL-1β (RAB0274) which were purchased from Sigma‑Aldrich (St. Louis, MO, USA).
RT‑qPCR
TRIzol (Invitrogen, Carlsbad, CA, USA) and mirVana kit (Applied Biosystems, Thermo Fisher Scientific, CA, USA) were used for the extraction of RNA in the detection of RNA and miRNA, respectively. TaqMan Gene Expression Assays kit and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific, CA, USA) were applied for the reverse transcription of RNA and miRNA, respectively. RT-qPCR reactions were carried out with the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The expression level of miR-103a-3p and the mRNA levels of IL-6, TNF-α and IL-1β were normalized to U6 and GAPDH, respectively. The data was analyzed with the quantification 2-ΔΔCq method (32).
Western blotting
The Ana-1 murine macrophages were first subjected to RIPA (Roche Diagnostics, Basel, Switzerland) for the extraction of total proteins. Then, the proteins were subjected to electrophorese on 8% SDS-polyacrylamide gels and transferring to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked by 5% non-fat milk at room temperature for 1 h, and incubated with primary antibodies against GAPDH (#5174, 1: 1000), PTEN (#9188, 1: 1000), p-PI3K (#17366, 1: 1000), PI3K (#4249, 1: 1000), p-AKT (#4060, 1: 1000), AKT (#4685, 1: 1000), NF-κB p-p65 (#3033; 1: 1,000) and NF-κB p65 (#8242; 1: 1,000), which were purchased from Cell Signaling Technology (Boston, MA, USA) at 4˚C overnight. GAPDH served as an internal control. The membranes were incubated with anti-rabbit IgG (7074, 1:1,000, Cell Signaling Technology, Boston, MA, USA). The protein bands were visualized by the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, USA). Densitometric analysis was performed with Quantity One 4.62 (Bio-Rad).
Dual luciferase reporter assay
miR-103a-3p was first predicted to target PTEN 3’UTR by TargetScan version 7.1 (http://www.targetscan.org/vert_71/).
Thereafter, PTEN wild-type (WT) 3’UTR containing complementary sequences for the seed sequence of miR-103a-3p was amplified by PCR and cloned into the psi-CHECK-2 Vector (Promega, Madison, MI, USA), while a mutant type (MUT) 3’UTR of PTEN was generated by QuikChange II Site-Directed Mutagenesis Kit (Stratagene, USA). The promoters of miR-103a-3p were cloned and inserted into the upstream of luciferase gene in the pGL3-Basic vector (Promega).
As for luciferase activity, the Ana-1 murine macrophages were plated into 96-well plates and co-transfected with 400 ng of either psi-CHECK-2-PTEN-WT-3’UTR or psi-CHECK-2-PTEN-MUT-3’-UTR and 50 ng antagomiR-103a-3p or antagomiR-NC by Lipofectamine 2000 (Invitrogen). At 48 h later, the firefly luciferase activity was detected by the dual luciferase assays system (Promega). And Renilla luciferase activity served as the internal control.
Statistical analysis
Data were analyzed by GraphPad Prism software version 5.04 (San Diego, CA, USA). The differences between two groups were analyzed by student's t-test, while the differences among 4 groups were analyzed by one-way analysis of variance followed by Bonferroni's post hoc test. Data were presented as the mean ± standard error of the mean. Experiments were performed at least 3 times. P<0.05 indicated a statistically significant difference.