Preparation of CSE
CSE was prepared as previously reported [26]. Briefly, the smoke of a 3R4F Research Cigarette (University of Kentucky, USA) was bubbled into a flask containing 10 mL of warm (37 °C) RPMI-1640 medium by use of a vacuum pump at a constant speed (each cigarette was smoked for 5 min). The CSE solution was adjusted to pH 7.4 and then sterilized by filtration through a 0.22-µm pore filter (Schleicher & Schuell GmbH, Dassel, Germany). For quality control, the solution was standardized by monitoring the absorbance at 320 nm (A320) and 540 nm (A540). CSE quality was accepted if ΔOD (A320-A540) was between 0.9 and 1.2. The resulting CSE solution, regarded as 100% CSE, was diluted with medium and used in experiments within 1 hour.
Cell culture and treatment
Simian virus 40 (SV40)-transformed human bronchial epithelial cells, which are nontumorigenic and retain features of parent HBE cells, were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). They were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Life Technologies/Gibco, Grand Island, NY), 100 µg/mL streptomycin, and 100 U/mL penicillin (Life Technologies/Gibco, Gaithersburg, MD) under 5% CO2 at 37°C. Cells were passaged at a ratio 1:3 every 2 days. After reaching 70-80% confluence, the cells were washed with phosphate-buffered saline, grown in RPMI-1640 medium supplemented with 10% FBS, and exposed to 0, 1, 2, or 4 % CSE for 48 hours.
RNA preparation and reverse-transcriptase polymerase chain reaction (RT-PCR)
The nuclear and cytoplasmic fractions of cells were extracted by use of the PARIS Kit Protein and RNA Isolation System (Thermo Fisher Scientific). Total RNA (1 μg) was treated with 10 U of RNase R (Epicentre Technologies Corp., Madison, WI) in 1× RNase R reaction buffer in a total volume of 10 µl. The mixture was incubated at 37 °C for 1 hour. Total RNA (1 μg) was transcribed into cDNA by HiScript II Q RT Supermix (Vazyme Biotech, Nanjing, China). The polymerase chain reaction reactions (PCR) were evaluated by checking the PCR products on 2% w/v agarose gels. The primers are listed in Table 1.
Table 1 Primer sequences used
hsa_circ_0061052 5′-GGAGACAGGCATGGAGAAGA-3′
5′-GACCTGCACGGTCTCCTG-3′
GAPDH 5′-GGAGCGAGATCCCTCCAAAAT-3′
5′- GGCTGTTGTCATACTTCTCATGG-3′
Mouse GAPDH 5′-GTCTTCACTACCATGGAGAAGG-3′
5′-TCATGGATGACCTTGGCCAG-3′
miR-515-5p 5′-GGGTTCTCCAAAAGAAAGCAC-3′
5′-CAGTGCGTGTCGTGGAGT-3′
miR-1182 5′-GGGGAGGGTCTTGGGAGGGA-3′
5′-CAGTGCGTGTCGTGGAGT-3′
miR-1304-5p 5′-GGGTTTGAGGCTACAGTGA-3′
5′-CAGTGCGTGTCGTGGAGT-3′
miR-136-5p 5′-GGGACTCCATTTGTTTTGAT-3′
5′-CAGTGCGTGTCGTGGAGT-3′
U6 5′-CGCTTCGGCAGCACATATACTAAAATTGGAAC-3′
5′-GCTTCACGAATTTGCGTGTCATCCTTGC-3′
Quantitative real-time PCR
Total cellular RNA was isolated by Trizol (Invitrogen) according to the manufacturers' recommendations. To assess the circRNAs and the miRNAs, 1 mg of total RNA and HiScript II Q Select RT Supermix (Vazyme biotech) were used in reverse transcription according to the manufacturer's protocol. GAPDH RNA was used as a control. The primers are listed in Table 1. Quantitative real-time PCR was performed with Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and a LightCycler 96 instrument (Roche, Swiss). The fold change in the expression of each gene was calculated by the threshold cycle (Ct) method using the formula 2-(ΔΔCt)[27].
Western blots
Proteins extracted from cultured cells or lung tissues of mice (16 male BALB/c mice (age 6-8 weeks)) were quantified with BCA protein assay kits (Beyotime, China). Equal amounts (80 μg) of protein were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Membranes were then incubated overnight at 4 °C with a 1:1000 dilution of anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH, Sigma) and an antibody for E-cadherin, N-cadherin, vimentin (Cell Signaling Technology, Beverly, MA), α-smooth muscle actin (α-SMA, Abcam), or FoxC1(Abcam). After additional incubation with a 1:1000 dilution of an anti-immunoglobin horseradish peroxidase-linked antibody for 1 h, the immune complexes were measured by enhanced chemiluminescence (Cell Signaling Technology). For densitometric analyses, protein bands on the blots were assessed by Image J software.
Fluorescence in situ hybridization assay (FISH)
FISH assays were performed with Fluorescence In situ Hybridization Kits (RiBoBio, Guangzhou, China). Briefly, cells grown on coverslips were fixed with 4% paraformaldehyde at room temperature for 10 min and treated with 0.5% Triton X-100 at 4 °C for 5 min. The samples were treated with pre-hybridization buffer at 37 °C for 30 min and then in hybridization buffer at 37 °C for 12–16 hours with a Cy3-labeled circRNA probe (Ribobio, Guangzhou, China) in a humid and dark environment. The samples were mounted with fluorescence mounting medium and imaged by use of a microscope (Zeiss, LSM700B, Germany).
Cell transfection
An miR-515-5p mimic, an miR-515-5p inhibitor, an miRNA negative control mimic (con mimic), an miRNA negative control inhibitor, hsa_circ_0061052 siRNA, and a control siRNA were synthesized by RiBoBio (Guangzhou, China). Cells were transiently transfected by use of Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. At 24 hours after transfection, cells were harvested and used for experiments.
Luciferase reporter assay
The binding of hsa_circ_0061052 to miR-515-5p and miR-515-5p to target genes was determined by luciferase-reporter assays. Either of two forms of hsa_circ_0061052 (wt-hsa_circ_0061052 or mut-hsa_circ_0061052) was cloned into a psiCHECK2 vector, and either of two forms of the FoxC1 3′-UTR (wt-FoxC1 3′-UTR or mut-FoxC1 3′-UTR) was cloned into a psiCHECK2 vector (Genechem Co., Ltd. Shanghai, China) and transfected into HBE cells. Luciferase activity was determined with Dual Luciferase Reporter Gene Assay Kits (Beyotime) according to the manufacturer's protocol. The Renilla luciferase activity was normalized by firefly luciferase.
Mice exposed to CS
Male BALB/c mice at 6-8 weeks of age were purchased from Animal Core Facility of Nanjing Medical University and housed in Jiangsu Province Medicine, Pesticide and Veterinary Drug Safety Evaluation and Research Center. Animals were treated humanely and with regard for alleviation of suffering according to a protocol approved by the Nanjing Medical University Animal Care and Use Committee.
To observe the effects of CS on airway obstruction of lungs, 16 male BALB/c mice (age 6-8 weeks) were divided into four groups: normal control and low-, medium-, and high-CS exposure groups. The low-, medium-, and high-CS groups were exposed in a whole-body exposure system (Beijing Huironghe Technology CO., Ltd., China) to CS from 3R4F Research Cigarettes (University of Kentucky, USA) at concentrations of 0, 100, 200, or 300 mg/m3 total particulate matter (TPM) for 60 min twice a day, 4 hours apart, 5 days a week for a total of 16 weeks. Humidity, temperature, and O2 level in the chamber were measured continuously during the exposure period. In the first week, there was increasing exposure, as follows: mice were placed in the chamber and exposed for 20 min on the first day, 30 min on the second day, and 60 min on the third day until the end. Age-matched mice kept in a similar environment without exposure to CS served as controls. Experiments were accomplished with n = 4 randomized animals per group.
Lung function measurement
For mice, airway hyper-responsiveness (AHR) was measured as the change in airway function by use of whole-body plethysmography (Buxco Electronics Ltd., USA), as previously reported [26]. Individual mice were placed unrestricted in a chamber connected to a pressure transducer to measure pressure changes inside the chamber. After acclimation, methacholine (0, 12.5, 25, or 50 mg/mL) were nebulized for 2 min, and enhanced pause (Penh) was recorded during the response period using FinePoint software (Buxco Electronics Ltd., USA). Penh, a dimensionless unit, correlates with pulmonary resistance. Values were averaged and expressed as absolute Penh values.
Masson trichrome staining
The lungs of mice were fixed with 4% paraformaldehyde and embedded in paraffin. For histological analysis and detection of collagen deposition, successive 5-µm lung sections were placed on slides and subjected to staining with trichrome (Masson) kits (Sigma-Aldrich, Germany), according to the manufacturer's instructions. Collagen content was determined by the ratio of collagen surface area (blue) to total surface area (red). Image J software was used to evaluate collagen deposition.
Immunohistochemistry (IHC)
Mouse lungs were fixed with 4% paraformaldehyde and embedded in paraffin. To evaluate α-SMA expression, consecutive 5-μm lung sections were placed on glass slides and stained with an α-SMA stain according to the manufacturer's instructions (Solarbio Life Science, China). An IHC scoring (IRS) system was used to quantify IHC staining. The percentage of positively stained bronchial epithelial cells was as follows: 1 (<10%), 2 (10-50%), 3 (50-75%), and 4 (> 75%). Staining intensity was 0-3: 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow-brown); 3, strong staining (brown). The staining index was obtained by multiplying the percentage of positive bronchial epithelial cells by the staining intensity, ranging from 0 to 12.
Statistical analyses
All experiments were performed in triplicate. Derived values are presented as means ± SD. Comparison of means among multiple groups was accomplished by one-way analysis of variance (ANOVA), and a multiple-range least significant difference (LSD) was used for inter-group comparisons. P values < 0.05 were considered statistically significant. All statistical analyses were performed with SPSS 18.0.