2.1 Clinical samples
In this study, a total of 30 children with asthma (18 males and 12 females, with an average age of 7.1 ± 3.1 years) and 30 healthy children (20 males and 10 females, with an average age of 7.7 ± 2.8 years) were recruited from Nanyang Central Hospital. Exclusion criteria were children having heart, liver, kidney, malignant hematological diseases, tumors diseases or other lung diseases. Blood samples were collected from children with asthma before treatment and health control during health examination, and plasma were isolated by centrifugation at 1000 g for 15 minutes and then stored at −80°C. This study was approved by the Ethnic Committee of Nanyang Central Hospital, and informed consent was obtained from each patient involved in this study.
2.2 Animals
Female BALB/c mice (6–8 weeks old, 20 ± 2 g) were obtained from the Laboratory Animal Center of Zhengzhou University. Mice were maintained in sterile cages under standard conditions (12 h light/dark cycle; temperature 22-25°C; humidity, 55-60%) and free access to standard pellet food and water. All animal experiments in this study were approved by the Animal Ethics Committee of Nanyang Central Hospital.
2.3 Cell lines and culture
The human bronchial epithelial cell line BEAS-2B was obtained from ATCC (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA), 100 U/mL penicillin and 100 µg /mL streptomycin (Sigma, St. Louis, MO, USA), and maintained with 5% CO2 at 37°C. When cells were grown to over 80% confluence, 1 µg/mL LPS (Sigma, St. Louis, MO, USA) was added to the BEAS-2B cell medium and incubated for 24 h.
2.4 Cell transfection
Overexpression plasmids of lncRNA CDKN2B-AS1 (pcDNA-CDKN2B-AS1), ZFP36 (pcDNA-ZFP36), small interfering RNAs targeting CDKN2B-AS1 (si-CDKN2B-AS1) and ZFP36 (si-ZFP36), and their corresponding negative controls (vector and scramble) were obtained from RiboBio (Guangzhou, China). Cells were seeded in 6-well plates at a density of 2×105 cells/mL, and when reaching 70% confluence, cell transfection were performed by using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Cells were collected after transfection for 48 h for further experiments.
2.5 RNA extraction and RT-qPCR
The total RNA in plasma were extracted by using QIAamp RNA Blood Mini Kit (Qiagen, Hilden, Germany), and the total RNA of BEAS-2B cells or tissues was extracted by using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cDNA synthesis was performed by using a Prime Script RT reagent Kit (Takara, Dalian, China). RT-qPCR were conducted with SYBR Premix Ex Taq II (Takara, Dalian, China) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) under the following conditions: 95°C for 1 min, 35 cycles of 95°C for 20 s, 56°C for 10 s and 72°C for 15 s. PCR reaction system contained 12.5 µL of SYBR Premix Ex Taq Ⅱ, 1.0 µL of RT primer, 1 µL of cDNA sample, and 10.5 µL of double distilled H2O. Relative gene expression was calculated normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and calculated by 2−ΔΔCT method. Primers were as follows: lncRNA CDKN2B-AS1 (forward: 5’-TGC TCT ATC CGC CAA TCA GG-3’, reverse: 5’-GGG CCT CAG TGG CAC ATA CC-3’), GAPDH (forward: 5’-CTG GGC TAC ACT GAG CAC C-3’, reverse: 5’-AAG TGG TCG TTG AGG GCA ATG-3’).
2.6 CCK-8 assay
Cell viability was measured by using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. Briefly, after treatment, BEAS-2B cells (1×104 cells/well) was seeded into 96-well plates and cultured for 48 h. Then 10 µL of CCK-8 solution was added to wells and incubated for 2 h at 37℃. The absorbance of each well was measured at 450 nm with a Microplate Reader (Bio-Rad, Hercules, CA).
2.4. Cell apoptosis analysis
BEAS-2B cells were collected and stained with the Annexin V-FITC/PI apoptosis detection kit (BD Bioscience, San Jose, CA, USA). Briefly, cells were resuspended in 1× binding buffer (1 × 106 cells/mL). Then 5 µL Annexin V-FITC and 5 µL PI were added into cell suspension and incubated for 15 min in the dark at room temperature. Cell apoptosis was detected by using Flow Cytometer (BD Bioscience, San Jose, CA, USA) according to the manufacturer's instruction.
2.5 Enzyme-linked immunosorbent assay (ELISA)
Following the indicated treatments, the supernatant of BEAS-2B cells or BALF were collected, and then the concentration of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were determined with the commercial ELISA kits purchased from R&D systems according to the manufacturer’s instructions.
2.6 Western blot analysis
Proteins were extracted from BEAS-2B cells and quantified using the BCA method (Millipore, Billerica, MA, USA). Then equal amount of protein was subjected to 10% SDS-PAGE at 70 V for 30 min then 120 V for 90 min. And the protein bands were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) at 300 mA for 2 h. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4 ºC with the following primary antibodies obtained from Abcam (Cambridge, UK): rabbit polyclonal anti-ZFP36 antibody (1:1000, ab83579), rabbit polyclonal anti-NR4A1 antibody (1:500, ab13851), rabbit monoclonal anti-p65 antibody (1:1000, ab32536) and rabbit polyclonal anti-GAPDH antibody (1:2500, ab9485), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000, ab6721) for 1 h. Subsequently, the protein bands were visualized with ECL detection reagents and analyzed with ImageJ software (National Institutes of Health, Bethesda, MA, USA).
2.7 Methylation-specific PCR (MSP)
MS-PCR was used to detect the methylation status of the ZFP36 promoter. Cell DNA extraction was conducted with the genomic DNA extraction kit (Tiangen Biochemistry Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The DNA concentration was determined using a UV spectrophotometer. The extracted DNA (10 µg) was added with 5.5 µL of 3 mol/L NaOH to denature at 37°C for 10 min. Next, DNA was added with 20 µL of 10 mM hydroquinone and 520 µL of 40.5% sodium hydrogen sulfite, and then covered by 200 µL mineral oil. Then the modified DNA was purified by wizard DNA column. The purified solution was added with 5.5 µL of 3 mol/L NaOH to denature for 10 min, and then 5.5 µL of 3 M sodium acetate and 120 µL cold absolute ethanol were added to precipitate and recycle DNA. MS-PCR reaction was conducted using ABI7500 quantitative PCR instrument (ABI Company, Oyster Bay, NY). The reaction products were then analyzed by agarose gel electrophoresis and imaged with a gel electrophoresis imaging analysis system.
2.8 Chromatin immunoprecipitation (ChIP)
EZ-Magna ChIP TMA kit (Millipore, Billerica, MA) were employed for chromatin immunoprecipitation (ChIP) analysis following the manufacturer’s guideline. Briefly, BEAS-2B cells were cross‐linked with 1% formaldehyde for 10 min, and then 125 mM glycine was added to terminate the crosslinking. Next, cells were lysed in lysis buffer, and chromatin fragments at 200‐1000 bp were obtained by cracking the cells through ultrasound. The supernatant was centrifuged and the fragments were collected in three tubes, which were supplemented with the target protein specific antibody (DNMT1, ab13537, Abcam) or the negative control antibody (IgG, ab10948, Abcam) for incubation at 4°C overnight. The DNA-protein complex was precipitated with Protein Agarose/Sepharose. The precipitated DNA fragments were purified and subjected to RT-qPCR analysis.
2.9 RNA immunoprecipitation (RIP) assay
The binding of lncRNA CDKN2B-AS1 with DNMT1 was determined by using the Magna RIP RNA-Binding Protein Immunoprecipitation kits (Merck Millipore, Billerica, MA). Briefly, cells were lysed by using a RIPA lysate buffer (Beyotime Biotechnology Co., Shanghai, China) for 5 min, and the supernatant was collected by centrifugation at 4°C. Subsequently, 50 µL of protein A/G‐beads was resuspended with 100 µL of RIP wash buffer, and 5 µg of anti‐DNMT1 antibody (ab13537, 1:100, Abcam) or NC antibody (IgG, ab10948, 1:100, Abcam) were added and incubated for 30 min at the room temprature. After washed, the protein A/G‐bead‐antibody complexes were resuspended with 900 µL of RIP wash buffer and incubated with 100 µL of supernatant overnight at 4°C. After immunoprecipitation, the protein A/G‐beads were collected and washed with RIP wash buffer and deposited 5 times. Finally, the protein A/G‐bead‐protein complexes were blended with proteinase K to extract RNA. Relative RNA expression was analyzed with RT‐qPCR.
2.10 Co-immunoprecipitation (Co-IP) assay
BEAS-2B cells were lysed RIPA buffer (Beyotime, Shanghai, China), and the supernatant was collected and incubated with ZFP36 antibody at 4°C overnight. Then the mixture was incubated with 100 µL of protein A/G agarose beads (Takara Biotechnology, Dalian, China) overnight at 4℃. Subsequently, the agarose beads-antigen-antibody complex was collected by instantaneous centrifugation and washed with PBS for three times. Next, the complex was boiled with protein loading buffer for 5 min. The supernatant was collected by centrifugation and analyzed by using Western blot to detect the expression of interaction proteins.
2.11 Animal experimental protocols
BALB/c mice were randomly divided into three groups (n=8 per group): control group, OVA group, and OVA+si-CDKN2B-AS1 group. On day 1 and 14, the mice were sensitized with 20 µg of ovalbumin (OVA) with 2 mg aluminum hydroxide adsorbed in 200 µL of PBS by intraperitoneal injection. From day 21 to 23, mice were challenged by intranasal inhalations of 100 µg OVA adsorbed in 20 µL of PBS once a day. For si-CDKN2B-AS1 treatment, 100 µg of si-CDKN2B-AS1 was administered daily via intraperitoneal injection on day 21 to 23. For NC group, mice were treated with an equal volume of PBS by using the same method. The mice were euthanized 24 h after the last challenge.
2.12 Measurement of Airway Hyperresponsiveness
Airway hyperresponsiveness (AHR) was detected by using noninvasive whole-body plethysmography (Model PLY 3211; Buxco, Sharon, CT, USA). Within 24 h following the final OVA challenge, the mice in all groups were treated with 0, 5, 10, 25 and 50 mg/mL methacholine aerosol for 3 min, followed by 2 min of rest, and the enhanced pause (Penh) value of unrestrained mice within 5 min was recorded.
2.13 Collection of bronchoalveolar lavage fluid (BALF) and cell counting
After mice were anesthetized, the tracheas were cannulated and lavaged with 0.8 mL aliquots of cold PBS twice to collect bronchoalveolar lavage fluid (BALF). The BALF samples were immediately centrifuged and kept at -80℃. The cell pellets from BALF were resuspended in PBS (0.5 mL). Total cell counting was done with a hemocytometer. And the Kwik-Diff staining set (Thermo, USA) was used for counting of differential cell counts (eosinophils, macrophages, neutrophils, and lymphocytes) in BALF according to the manufacturer’s instructions.
2.14 Histopathological analysis
Lung tissues were collected and fixed in 10 % neutral buffered formalin, and then embedded in paraffin and sliced. Paraffin sections were stained with hematoxylin and eosin (H&E) using a standard protocol and analyzed by light microscopy.
2.15 Measurement of the level of OVA-specific IgE in serum
On day 24 after the last OVA challenge, mice were euthanized and the blood samples were collected by puncturing the vena cava. The blood samples were centrifuged at 1000 g for 10 min to obtain serum samples. The level of OVA-Specific immunoglobulin E (IgE) in serum was measured by ELISA kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions.
2.16 Statistical analysis
Data analysis was performed by SPSS version 22.0 software. Experimental results from three times independent experiments were presented as mean± standard deviation (SD). Comparisons between two groups or multiple groups were performed by using student’s t-test or analysis of variance (ANOVA), respectively. P<0.05 was considered to be statistically significant.