In silico analyses
Ten genes exhibiting the strongest interaction with PM2.5 were achieved from the Comparative Toxicogenomic Database (CTD, http://ctdbase.org). The potential miRNAs targeting HMOX1 gene were predicted using TargetScan (www.targetscan.org), miRTar (mirtar.mbc.nctu.edu.tw), Starbase (starbase.sysu.edu.cn/), DIANA (diana.imis.athena-innovation.gr/DianaTools/), respectively. RNAhybrid algorithm (bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) was used to predict the free energy of potential miRNA: mRNA duplexes.
Cell culture, transfection, and PM2.5 treatment
Human lung cancer cell line H226 and embryo kidney cell line HEK293T were obtained from American Tissue Type Culture Collection (ATCC, Manassas, VA). Human lung normal epithelial cell line (HBE) is a gift from professor Wen Chen (Sun Yat-Sen University, Guangzhou). H226 was cultured in RPMI-1640 medium, HEK293T was cultured in DMEM medium, and HBE was cultured in MEM medium plus 10% FBS, respectively. The cells were maintained at 37◦C in a humidified 5% CO2 incubator.
Multiple commercially obtained miRNA mimics (Ruibo, Shanghai, China), miRNA inhibitor (IDT, Coraville, IA), and siRNAs targeting YBX1 and HMOX1 (Ruibo, Guangzhou, China) were transfected into cells using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA), while constructed plasmids were transfected into cells by Lipofectamine 2000 (Invitrogen).
Ambient PM2.5 were collected from Shijiazhuang, China, and the details about the collection, sample extraction and components analyses were presented in our previous study . PM2.5 was dissolved in DMSO to produce 100mg/ml stock solutions, and then added to the cell culture at the final concentration of 25μg/ml, 50μg/ml, and 100μg/ml, respectively. HBE cells treated with an equal amount of 0.1% DMSO were used as solvent control.
Fluorescent-based RNA electrophoretic mobility shift assay (FREMSA)
All oligonucleotides and primers used in this study were purchased from Sangon Biotech (Shanghai, China). The dye-hsa-miR-760 oligonucleotide was labeled with IRDye® 800 dye on its 5’ end, while the cognate HMOX1 mRNA FREMSA was performed according to the protocol described in our previous study . Briefly, Briefly, both miRNA and mRNA oligonucleotides were heated for 2 minutes at 95℃ to relax RNA secondary structures and immediately placed on ice. And then, 400 nM synthetic miRNA or/and 400nM cognate mRNA oligonucleotides were mixed with basic reaction buffer (1x Binding Buffer, 5% glycerol, 200 mM KCl, and 100 mM MgCl2). All reaction mixtures were cultured at 25℃ for 20 min to form miRNA:mRNA duplex. The reaction mixtures were then separated on a 12% PAGE by electrophoresis at 4℃, and the resultant mobility shifts were scanned by Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoin, NE).
MicroRNA pull-down assays and MS analysis
The miRNA pull-down experiment was carried out as described in the previous study . Briefly, a biotin molecule was covalently attached to the 3′ end of the mature hsa-miR-760 or negative control strand. Modified biotin-miR-760 mimics and negative control were transfected into the HBE cells. Cells were harvested at 24h after transfection, and lysed using the lysis buffer that containing 3% IGEPAL ® CA-630 (Sigma-Aldrich, St. Louis, MO), protease inhibitor cocktail (Roche, Basel, Switzerland), and RNaseOUT (Thermo scientific, Tewksbury, MA). The cytoplasmic lysate was mixed with precoated beads (Life technologies) and incubated (with rotation) at 4 °C overnight to obtain miRNA-mRNA-protein complex. The harvested complex was then divided into two parts. One portion was used to isolate RNA using Trizol (Life Technologies, Carlsbad, CA), while the other portion was used to analyze proteins based on LC-MS/MS system by Applied Protein Technology (Shanghai, China).
Dual-luciferase reporter gene assay
Hsa-miR-760 was predicted to target both the 3’UTR and coding region of the HMOX1 gene. The core HMOX1 3’-UTR that harboring the hsa-miR-760 response element was chemically synthesized and subcloned into the psiCHECK2 reporter vector, to detect the potential interactions between hsa-miR-760 and HMOX1 3’UTR. We further modified the psiCHECK2 vector to express the luciferase-HMOX1 fusion protein, by deleting a “T” nucleotide from the stop codon “TAA” of Renilla luciferase gene based on the change mutation method. Then the coding region of HMOX1 that containing the response elements of hsa-miR-760 was subcloned into the modified psiCHECK2 vector. Specific mutations in the target sites matching to hsa-miR-760 seed sequence were introduced by PCR-based site-specific mutagenesis.
For the luciferase assays, HBE and 293T cells were seeded at 60% confluency in 96-well plates and co-transfected by constructed plasmids (100 ng/well) together with hsa-miR-760, or negative control (NC) mimics (100 nM) using lipo2000. Cells were harvested at 48h after transfection, and the luciferase activity was measured using the dual-luciferase kit (Promega, Madison, WI).
RNA isolation and quantification
All mRNA and miRNA primer sequences used in this study were listed in Supplementary Table 2. Both mRNAs and miRNAs were extracted from cells using TRIZOL, and cDNA was synthesized with a random primer or specific miRNA or stem-loop reverse transcription primers, using Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific). Quantitative Real-Time PCR (qRT-PCR) was conducted using SYBR Green Kit (Qiagen) on the Light Cycler 480 Analyzer (Roche). All of the reactions, including the no-template controls, were run in triplicate. The miRNA levels were normalized to U6 snRNA, while mRNA levels were normalized to GAPDH.
mRNA degradation assay
This assay is designed to measure the decay kinetics of mRNAs. HBE cells were transfected with hsa-miR-760 mimics or NC mimics, incubated for 24h, and exposed to actinomycin D (final concentration: 3μg/ml). The cells were then harvested at 0, 4, 8, and 16 h after actinomycin D treatment, respectively, and total RNAs were prepared to test the relative HMOX1 expression as mentioned above.
RNA immunoprecipitation (RIP)
RIP assay was conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA), according to the manufacturer’s protocols. Briefly, the cell lysate was incubated with antibodies against YBX1 and IgG control (Abcam, Cambridge, MA) overnight at 4 °C. The RNA-protein complex was recovered by Protein G Dynabeads and reverse cross-linked with proteinase K. Finally, the protein-binding RNAs were extracted using TRIZOL reagent and analyzed by qRT-PCR.
Western blot analysis
Total proteins were isolated from the cells using RIPA lysis buffer (Thermo Scientific). Antibodies against HMOX1 and GAPDH were purchased from Abcam, and antibodies against BCL2 and BAX were obtained from Cell Signal Technology (Danvers, MA). Western blotting assays were carried out using the Odyssey™ Western Blotting Kit (LI-COR Biosciences), and analyzed based on the Odyssey CLx Infrared Imaging System.
TUNEL Apoptosis Assays
Apoptosis was detected using the riboAPO™ One-Step TUNEL Apoptosis Kit (RiboBio, Guangzhou, China), according to the manufacturer’s protocol. In brief, cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with TdT Enzyme and TAM-dUTP Mixture protected from light, serially. The reaction was stopped by SSC, and the apoptotic cells stained by TUNEL were observed under a fluorescence microscope.
Cells were treated with PM2.5 as indicated and then exposed to membrane permeable fluorescent probe DCFH-DA (final concentration: 10 mM). After incubation for 20min at 37°C, the cells were harvested and resuspended in PBS. Fluoresce signaling was detected by Beckman Coulter (USA).
The quantitative variables are exhibited as mean ± standard deviation (SD). Statistical analysis was conducted using SPSS Statistics Version 21.0 (SPSS Inc., Chicago, IL). Student’s t test was used to compare the differences in luciferase reporter gene assays, while one-way ANOVA on ranks test was used to detect the differences between subgroups for protein or RNA levels. Differences were regarded as statistically significant when P < 0.05.