4.1. MG Culture
The MG virulent strain used in this study was MG-HS strain isolated from henneries in Hubei, China [52] and was donated by the State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University (Wuhan, China). MG-HS was cultured at 37 ℃ in modified FM-4 medium supplemented with 12% (v/v) porcine serum and 10% yeast extract until the mid-log phase. The concentration of MG-HS was determined by the acid-mediated shift of phenol red dye from red to orange as previously described. The number of viable Mycoplasmas in a suspension was then determined by a color-changing unit (CCU) assay.
4.2. Infection Experiments
One hundred embryos of White Leghorn specific-pathogen-free (SPF) chickens were incubated on the ninth day and the all were injected with 300 µL of MG-HS at 10 CCU/mL. Other 100 chicken embryos were injected with the same dosage of the diluent to serve as controls. The viability of the chicken embryos was examined by eye under a candling machine. The dead embryos were eliminated. The mortality rates of the chicken embryos of the infection and control groups were 12.3% and 7%, respectively. Whole-lung tissue samples from six infected live chicken embryos and six controls were collected on days 6, 10, and 11 post-infection and stored in RNA fixer (BioTeke Co., Ltd., Beijing, China).
4.3. Cell Culture and Treatment
The chicken embryonic fibroblast cell line (DF-1) was obtained and authenticated from American Type Culture Collection (Rockville, MD, USA). Cells were maintained in Dulbecco’s modified eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) and 1% penicillin-streptavidinglutamine (PSG, Invitrogen, USA). Cells were incubated at 39 °C in a 5% CO2 humidified atmosphere. For transient transfection, DF-1 cells were plated evenly in 6-, 24- or 96-well plates and grown to 60% confluency without antibiotics and subsequently transfected with RNAs and/or plasmids using Lipofectamine 3000 (Invitrogen, USA). After 48 h, the cells in different groups were collected for further use. For MG-HS infection experiments, at 24 h post-transfection, cells were infected with MG-HS at the log phase (1 × 1010 CCU/ml) for the times mentioned in the figure legends.
4.4. microRNA Target Prediction and Sequences
Putative miR-181a-5p target genes were identified using a miRNA database (http://www.mirbase.org/) and target prediction tools: miRDB (http://www.mirdb.org/miRDB/), PicTar (http://pictar.mdc-berlin.de/), and TargetScan (http://www.targetscan.org/). The conservation of the target gene was analyzed by TargetScan (http://www.targetscan.org/). The duplex and mfe between miR-181a-5p and the 3’-UTR of the potential targets were analyzed by RNAhybrid (https://bibiserv.cebitec.unibielefeld.de/rnahybrid/). AmiGO (http://amigo.geneontology.org) was used to analyze the functions of the target genes of gga-miR-181a-5p in Gallus gallus.The sequences of all of the primers used in this study are shown in Table 1. All RNA oligonucleotides were designed and synthesized by GenePharm (Shanghai, China) and are shown in Table 2.
Table 1. Sequences of DNA primers.
Name
|
Primer Sequence (5′-3′)
|
Accession No.
|
|
Primers for CDS Cloning
|
|
PPM1B-CDS-F
|
CGCGGATCCATTGCATAAACATGGGTGCATTT
|
NM_001031052.1
|
PPM1B-CDS-R
|
GGAATTCCTACCACGGATCTTCTAGATCTCCA
|
|
|
Primers for 3′-UTR Cloning
|
|
PPM1B 3'UTR-F
|
TTCCTCGAGATGGGCATGGTACAGAGTGG
|
NM_001031052.1
|
PPM1B 3'UTR-R
|
AATGCGGCCGCTGCTATGAGAGAGCGTGTGG
|
|
Mut-PPM1B 3'UTR-F
|
GGAGGGGAAACAATACGTGTATCCATCTGTACATGATTTACATGCTGTG
|
NM_001031052.1
|
Mut-PPM1B 3'UTR-R
|
ATCATGTACAGATGGATACACGTATTGTTTCCCCTCCCAATAGCTTTTACT
|
|
|
Primers for RT-qPCR
|
|
GAPDH-F
|
GAGGGTAGTGAAGGCTGCTG
|
NM-204305
|
GAPDH-R
|
CACAACACGGTTGCTGTATC
|
|
PPM1B-F
|
AATCTGGGATGTAATGAGCAACG
|
NM_001031052.1
|
PPM1B-R
|
TCCAAGTGCTTATCCAACTCTGC
|
|
MAP3K7-F
|
GAGATCGAAGTGGAAGAGGT
|
XM_015284677.2
|
MAP3K7-R
|
TGGGTTTAGACAGGCTCCAT
|
|
IKBKB-F
|
CTGCCCGTGATGTTCCTGA
|
NM_001031397.1
|
IKBKB-R
|
TGAGAGCAGAGGCAATATCAGAC
|
|
NF-κB-F
|
GCCAGGTTGCCATCGTGT
|
NM-205129
|
NF-κB-R
|
CGTGCGTTTGCGCTTCTC
|
|
TLR-2-F
|
CGCAAGCTTATGTTCAACCAAAG
|
NM_001161650.1
|
TLR-2-R
|
CGCCTCGAGCTATGACTTCAAGG
|
|
MYD88-F
|
TCAGTTTGTCCAGGAGATG
|
NM-001030962
|
MYD88-R
|
GGTGTAATGAACCGCAAGATA
|
|
TNF-α-F
|
GGACAGCCTATGCCAACAAG
|
XM-015294124
|
TNF-α-R
|
ACACGACAGCCAAGTCAACG
|
|
IL-1β-F
|
ACTGGGCATCAAGGGCTACA
|
NM_204524.1
|
IL-1β-R
|
GCTGTCCAGGCGGTAGAAGA
|
|
PMGA1.2-F
|
TCATATAAAAACTTAATGGCTACTC
|
AF275312_2
|
PMGA1.2-R
|
CTCTTCTGTGATTGTATTAGCAG
|
|
RT-gga-miR-181a-5p-F
|
CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACTCACCG
|
MIMAT0001168
|
gga-miR-181a-5p-F
|
TAGGAACATTCAACGCTGTCGGT
|
|
gga-miR-181a-5p-R
|
ACTGGTGTCGTGGAGTCGGC
|
|
gga-5s-rRNA-F
|
CCATACCACCCTGGAAACGC
|
|
gga-5s-rRNA-R
|
TACTAACCGAGCCCGACCCT
|
|
RT-5S
|
AACTGGTGTCGTGGAGTCGGC
|
|
Table 2. Sequences of RNA oligonucleotides.
Name
|
Sequences (5′–3′)
|
gga-miR-181a-5p mimics
|
AACAUUCAACGCUGUCGGUGAGU
|
UCACCGACAGCGUUGAAUGUUUU
|
gga-miR-181a-5p NC
|
UUCUCCGAACGUGUCACGUTT
|
ACGUGACACGUUCGGAGAATT
|
gga-miR-181a-5p inhibitor
|
ACUCACCGACAGCGUUGAAUGUU
|
gga-miR-181a-5p inhibitor-NC
|
CAGUACUUUUGUGUAGUACAA
|
si-gga-PPM1B
|
GCAGGATCTCGTGTTGCAA
|
4.5. Primary CP-II cells culture
CP-II cells were isolated according to our established method [53]. Briefly, it was prepared from lung tissue of 15 day SPF chicken embryos. The lung tissue was cut, washed three times with Hank's solution; then, 0.25% trypsin was added, each embryo was added in an amount of 1 ml, digested in a water bath at 37 °C for 10 minutes, centrifuged at 800 rpm/min for 10 minutes, and the supernatant was discarded; then, 0.1% IV collagenase was added, each embryo was added in an amount of 1 ml, digested in a water bath at 37 °C for 15 min, centrifuged at 800 rpm/min for 10 minutes, the supernatant was discarded, and a moderate volume of 10% FBS in DMEM complete medium was gently pipetted. The cells were mixed and filtered through a 75 μm mesh to a sterile plate for adherence. The unattached suspension was centrifuged at 1200 rpm/min for 5 min, resuspended and centrifuged, and repeated 3 times. After resuspending the cells in DMEM complete medium supplemented with 20% FBS, the cells were filtered through a 38 μm sieve into a culture flask, and the cells were cultured in a humidified incubator at 37 °C, 5% CO2 for 18 hours, and then the culture solution was changed. At this time, the adherent cells are chicken embryo type II epithelial cells.
4.6. Exosome isolation, identification and labeling
Exosomes were purified from the cell culture supernatant of CP-II cells. Prior to culture medium collection, CP-II cells were washed twice with PBS, and the medium was switched to exosome-free medium (ultracentrifugation at 100,000X g for 16 h at 4 ℃) upon MG stimulation. The cells were then cultured for 48 h. The supernatant was collected and went through sequential ultracentrifugation at 2000 X g for 30 min, 10,000X g for 30 min, and 100,000 X g for 70 min at 4 ℃. The exosomes were washed once with PBS at 100,000X g for 70 min and suspended for further characterization.
A transmission electron microscope (TEM, Thermo Scientific, Waltham, MA) was used to identify the form of the exosomes. Nanoparticle tracking analysis (NTA, Brookhaven, New York) was used to measure exosome diameter and particle number. The protein content was measured using BCA protein assay (Thermo Scientific, Waltham, MA), and exosome markers CD9 and CD63 were detected by western blot analysis.
Fluorescence labeling of exosomes was performed according to the protocol previously described [54]. The PKH26 kit was used according to the instruction manual (Sigma-Aldrich, San Louis, MO). The labeled exosomes were washed at 100,000 g for 1 h, and the exosome pellet was diluted in PBS and used for the uptake experiment.
4.7. Dual-Luciferase Reporter Assay
The psi−CHECK™-2 dual-luciferase reporter vector (Promega, Madison, WI, USA) harboring the wild-type and mutant PPM1B 3′-UTR, which were inserted into the Xho I and Not I restriction sites 3′ to the end of the Renilla gene, were used to check the effect of miR-181a-5p on Renilla activity. The psi−CHECK™-2 mutant PPM1B′-UTR construct was generated by inducing a point mutation using the overlap extension PCR method. DF-1 cells were seeded on 24-well plates at a density of 3 × 105 cells per well and cultured until the cells reached approximately 60% confluence. Cells were then transfected with 200 ng of the luciferase reporter plasmid and 10 pmol of miR-181a-5p, miR-181a-5p-NC, miR-181a-5p-Inh or miR-181a-5p-Inh-NC using Lipofectamine 3000 (Invitrogen, USA). At 48 h post-transfection, the cells were collected, the Firefly and Renilla luciferase activities were determined using EnSpire & Multimode Reader (PerkinElmer, Inc., Waltham, USA) according to the manufacturer’s protocol (Promega, USA). Three independent repeats were performed for all above transfection experiments.
4.8. RNA Extraction and Quantitative Real-Time PCR (qPCR)
Total RNA was extracted from the cultured cells and frozen lung tissue specimens of chicken embryos using the TRIzol reagent (Invitrogen). According to the manufacturer’s instructions, 1 µg RNA from each sample was used to synthesize cDNA using the Prime Script™ RT reagent kit with gDNA Eraser (TaKaRa, Tokyo, Japan). The real-time PCR was performed with TransStart Top Green qPCR SuperMix (TRANSGEN, Beijing, China) on a CFX96 or CFX384 Touch™ instrument (Bio-Rad, Hercules,CA, USA). The relative mRNA levels were calculated using the Bio-Rad, CR SuperMix (TRANSGEN, Beijing, China). The expression levels of miR-181a-5p and PPM1B were measured by qPCR. 5S-RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were used as internal controls, respectively. The expression of 5S-RNA and GAPDH are stable in the course of MG infection. The experiment was repeated three times, The primers are listed in Table 1.
4.9. Western blot analysis
Total proteins were extracted using RIPA lysis buffer, separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane (Roche Life Sciences, Switzerland). Membranes were blocked with 5% skimmed milk and incubated with the appropriate antibodies. The PPM1B antibody was obtained from Proteintech, and phosphorylated MAP3K7 and phosphorylated IKBKB antibodies acquired from Cell Signaling Technology (Danvers, MA, China). Primary antibodies were applied to the membrane, followed by horseradish-peroxidase-conjugated secondary antibodies. The antigen-antibody complex on the membrane was detected with enhanced chemiluminescence reagent (Thermo Scientific, Waltham, MA, China).
4.10. Cell Proliferation, Cell Cycle and Cell Apoptosis
Cell proliferation was determined using the Cell Counting Kit-8 according to the manufacturer’s protocol (CCK-8, DOJINDO, Shanghai, China). DF-1 cells were plated at a density of 8 × 103 cells/well in a flat-bottomed, 96-well cell culture plate and allowed to grow for 4 h at 39 ◦C 37℃ with 5% CO2. The DF-1 cells were then transfected with miR-181a-5p, miR-181a-5p-NC, miR-181a-5p-Inh, or miR-181a-5p-Inh-NC. At 24 h post-transfection, the cells then were infected with 8 µL of MG-HS strain at 1010 CCU/mL. At 24 h, 48 h, and 72 h post-transfection, 10 µL of the CCK-8 solution was added to each well of the plate, which was then incubated at 37 ℃ for 4 h. The infected-MG cells (denoted as miR-free MG + ) and the uninfected-MG cells (blank) were cultured in a sterile incubator to avoid MG contamination. The optical density at 450 nm of each well plate was measured using a microplate reader (Bio-Rad, Hercules, CA, USA).The cell cycle and cell apoptosis assay were performed in 6-well plates. The DF-1 cells were transfected with the indicated RNA oligonucleotides. At 24 h post-transfection, the cells were infected with 130 µL of MG-HS strain at 1010 CCU/mL. At 24 h post-infection, the cell cycle was analyzed with a flow cytometer, using the cell cycle detection kit (KeyGEN, Nanjing, China). Similarly, miR-free-MG+ and blank MG MGrly, miR-free-MG+ and blank MGank MGlow cytometer, using the cell cycle detection kit (t-transfection, the cells were infected with 130 µL of MG-HS strain at 1010 CCU/ch nufacturer’s protocol of the Annexin V-FITC Apoptosis Detection Kit (DOJINDO, Shanghai, China).
4.11. Statistical Analysis
SPSS software (SPSS 20.0, IBM, Armonk, NY, USA) was used for statistical analyses. All results are presented as the mean values the c. Statistical significance was determined by using one-way ANOVA or Student’s t-test, and p-values of < 0.01 or < 0.05 were considered the statistically significant difference between groups.