Bacterial strains and cell tradition
G. parasuis serovar 5 stain LZ, SS2 pressure QH, and S. aureus ATCC29213 have been preserved in our laboratory. G. parasuis was grown on Trypticase Soy Agar (TSA) or Trypticase Soy Broth (TSB) (OXOID, Basingstoke, UK) with the addition of 0.01% nicotinamide adenine dinucleotide (NAD) and 5% (v/v) inactivated bovine serum at 37 °C. SS2 was cultured on TSA or TSB with 5% (v/v) inactivated bovine serum at 37 °C. S. aureus was grown on Luria Broth medium.
PIEC cells and PK-15 cells have been preserved in our laboratory. PIEC cells have been cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, USA), and PK-15 cells have been cultured in DMEM medium (Gibco, Carlsbad, USA) supplemented with 10% FBS, 100 mg/mL streptomycin and 100 U/mL penicillin. All these cells have been maintained in a humidified incubator at 37 °C with 5% CO2.
Exosomes isolation
Ultracentrifugation is the gold customary for exosome isolation [25]. Exosomes have been remoted from the supernatant of PIEC tradition with exosome-depleted medium following the improved technique in line with the ultracentrifugation steps reported [26, 27]. Briefly, centrifugation at 500 × g for five min to take away cells from the samples, transferring the supernatant to a brand new polycarbonate tube and centrifuging 10 min at 2000 × g. The supernatant was collected and transferred to a brand new polycarbonate tube. Then, centrifugation for 30 min at 10,000 × g to eradicate shed microvesicles (sMV, 200–1000 nm). The supernatant was collected and filtered with 0.22 μm membrane filter (Merck Millipore, Darmstadt, Germany) and additional centrifuged at 100,000 × g for two h at 4 °C twice to pellet the exosomes. Lastly, exosomes have been resuspended in 50 µl to 100 µl phosphate-buffered saline (PBS) and saved at -80 °C for additional use.
Exosome identification and internalization
The 50 µl exosome samples have been loaded into nanoscale bronze grating after unfavorable staining by uranyl acetate, and the form and measurement of the exosomes have been noticed by transmission electron microscopy (TEM) (Tecnai G2, FEI, USA) at 120 kV. The dimensions distribution of the exosomes was characterised utilizing a Nanosight NS300 (NanoSight Ltd., Amesbury, UK) outfitted with a 405 nm laser to find out the dimensions and amount of particles remoted. A video of 60s length was taken with a body price of 30 frames/s, and particle motion was analyzed utilizing NTA software program (model 2.3; NanoSight Ltd.). The focus of exosomal proteins was analyzed not directly with the BCA Protein Assay Package (Thermo Fisher Scientific, USA), and exosomal protein markers CD63 (ABclonal, Wuhan, China), HSP70 (Proteintech, Chicago, USA) and unfavorable marker for exosome purity Calnexin (Proteintech, Chicago, USA) have been detected by Western blot. The inexperienced fluorescent dye PKH67 (Sigma, St.Louis, USA) was utilized to label exosomes remoted from the tradition medium of PIEC cells. After recipient PK-15 cells have been incubated with the dye for 12 h, laser confocal microscopy (Leica, SP8, Germany) was carried out to visualise PKH67-labelled exosomes in PK-15 cells. The detailed procedures have been carried out in line with the earlier report [10].
Cell remedy to inhibit exosome secretion
A pharmacological inhibitor of impartial sphingomyelinase-2 (nSMase) GW4869 may diminished ceramide formation [28]. 20µM GW4869 (MCE, New Jersey, USA) was used to deal with PIEC cells for two h to dam exosome formation, and exosomes have been remoted. The content material of C24:1 Ceramide (d18:1/24:1(15Z))(aladdin, Shanghai, China) in exosomes was analyzed by Excessive-performance LC/MS to verify that the secretion of exosomes by PIEC cells was diminished after GW4869 remedy in line with the earlier report [28]. On the similar time, the extent of exosome-derived circHIF1α was analysed by qRT-PCR.
CircRNA sequencing and evaluation
Complete RNAs of PIEC cells and PIEC cells contaminated with G. parasuis have been extracted with TRIzol reagent (Invitrogen, Carlsbad, USA) and quantified by the NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, USA), then 1–2 g complete RNA from every pattern was chosen for RNA sequencing library building. Picture evaluation and base calling have been carried out utilizing Solexa pipeline v1.8 (Off-Line Base Caller software program, v1.8). Sequence high quality was examined utilizing the FastQC software program (v0.11.7). The trimmed reads (trimmed 5’, 3’-adaptor bases utilizing Cutadapt (v1.17)) have been aligned to the reference genome utilizing Star software program. Again splice junction Reads detection and Reads depend statistics have been carried out by CIRCexplorer 2. The differential expression was calculated by R software program edgeR. PCA evaluation and correlation evaluation based mostly on gene expression stage, and additional knowledge mining evaluation corresponding to clustering of differentially expressed genes, GO perform significance enrichment evaluation, pathway significance enrichment evaluation, and so forth have been accomplished by the customized program (python/R/shell) of KangCheng Bio-tech (Shanghai, China).
Quantitative real-time PCR (qRT-PCR)
Complete RNA was extracted utilizing a TRIzol reagent. RNA focus was measured by NanoDrop 2000. The full RNA was synthesized into cDNA with PrimeScript RT Reagent Package (Takara, Dalian, China) in accordance with the producer’s protocols. The cDNA was amplified with TB Inexperienced Premix Ex Taq (Takara, Dalian, China) on a Bio-Rad CFX96 system (Bio-Rad, CA, USA). The expression of circRNAs and mRNA was decided by 2−∆∆CT and normalized by Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin. The primers within the research have been listed in Desk S1.
RNase R remedy
Complete RNA (2 µg) was incubated for 30 min at 37 °C with or with out 3 U/µg RNase R (Geneseed, Guangzhou, China), adopted by qRT-PCR evaluation.
Actinomycin D assay
PIEC cells have been transferred to 24-well plates, uncovered to 2 µg/ml actinomycin D (MCE, New Jersey, USA) and picked up on the indicated time factors. The steadiness of circRNA and their mRNA was analysed by qRT-PCR.
Isolation of cytoplasmic and nuclear fractionation
PIEC cells have been harvested and handled utilizing a Cytoplasmic & Nuclear RNA Purification Package (Norgen Biotek, Canada) in line with the producer’s directions. β-actin and U6 served as controls of cytoplasmic RNA and nuclear RNA, respectively. The expression of circRNA, β-actin, and U6 was decided by qRT-PCR. The primers are listed in Desk S1.
Co-culture assay
A complete of two.5 × 105 PIEC cells have been seeded within the higher chamber of the transwell membrane (Corning, USA), and 5 × 105 PK-15 cells have been seeded within the decrease chamber. The higher PIEC cells have been contaminated by G. parasuis (MOI = 10) after which cultured at 37 °C with 5% CO2 for 12 and 24 h, respectively. The qRT-PCR know-how was used to detect the adjustments of circHIF1α in higher cells (PIEC) and decrease cells (PK-15) and their supernatants, respectively.
Plasmid, siRNAs, and cell transfection
The complete size of the liner sequence of circHIF1α was amplified and subcloned into the lentiviral vector pLC5-ciR (Geneseed, Guangzhou, China) to assemble circHIF1α overexpression vector, named ov-circHIF1α. Two siRNAs concentrating on circHIF1α (si-circHIF1α), two siRNAs concentrating on IGF2BP3 (si-circHIF1α), and unfavorable management (si-NC) have been synthesized by Boshang (Jinan, China). FLAG-tagged expression pcDNA3.1 vectors for full-length swine IGF2BP3 and site-directed mutants have been supplied by Boshang (Jinan, China). Full-length circHIF1α and its truncations have been subcloned into pcDNA3.1 (Boshang, Jinan, China). Mobile transfection of siRNA and plasmid was performed utilizing Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, USA) and Lipofectamine™ 3000 package (Invitrogen, Carlsbad, USA) in line with the producer’s directions, respectively. The detailed oligonucleotide sequences used on this research are proven in Desk S2, and the detailed supply info for all plasmids is listed in Desk S3.
Cell proliferation and cell cycle assays
The expansion curves of PK-15 cells handled in 12, 24, 36, and 48 h have been obtained utilizing Cell Counting Package-8 (Beyotime, Shanghai, China) in line with the protocols of the producer. For the EdU assay package (Beyotime, Shanghai, China), the cells have been incubated with 1 × EdU working answer at 37 °C for two h, after which subsequent staining and visualization have been carried out in accordance with the producer’s directions. The cell proliferation price was calculated by the ratio of EdU-positive cells (purple) to DAPI-positive cells (blue). PK-15 cells have been collected and stuck in 70% chilly ethanol in a single day at 4 °C. Cell cycle distribution was carried out utilizing a circulate cytometer (LSR Fortessa, BD, USA) after cells have been handled with propidium iodide (PI, 50 µg/mL) and RNase A (100 µg/mL). Knowledge have been analyzed utilizing ModFit LT 5.0.
RNA pulldown assay
The complete size of circHIF1a was constructed on pCNDA3.1 vector and digested with FastDigest BamH I (Thermo Fisher Scientific, Waltham, USA), after which transcribed in vitro with MEGAscript RNA T7 Transcription Package (Invitrogen, Carlsbad, USA). The probe was ready by linking biotin with biotin 3’ Finish Desthiobiotinylation Package (Thermo Fisher Scientific, Waltham, USA) in line with the producer’s directions. RNA pulldown assay was carried out with Pierce™ RNA 3’ Finish Desthiobiotinylation Package (Thermo Fisher Scientific, Waltham, USA). The recovered protein was verified by Western blot or mass spectrometry assays.
RNA immunoprecipitation (RIP) assay
The RIP assay was carried out with PureBinding®RNA Immunoprecipitation Package (Geneseed, Guangzhou, China) to find out the interplay between circHIF1α and its interacting proteins. Briefly, 2 × 107 cells have been harvested and lysed by RIP lysis buffer, then incubated with magnetic beads conjugated with antibodies in opposition to IgG (Cell Signaling Know-how, Beverly, USA). The coprecipitated RNAs have been examined utilizing qRT-PCR with particular primers.
Methylated RNA immunoprecipitation (MeRIP) assay
The Methylated RNA Immunoprecipitation Package (BersinBio, Guangzhou, China) was utilized to look at the m6A modifications on circHIF1α on the idea of the producer’s directions. Briefly, the whole RNA was extracted by Trizol reagent after accumulating 2 × 107 PK-15 cells. The extracted RNA fragments have been remodeled into about 100 nt lengths, after which 6% RNA was used because the enter group. The remaining RNA was divided into two teams and incubated with m6A antibody and IgG antibody at 4 °C for 4 h, respectively. Protein A/G magnetic beads have been added to incubate with antibody at 4 °C for 1 h. The enriched RNA was extracted and detected by qRT-PCR.
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF)
A FAM-marked probe of circHIF1α was synthesized by BersinBio Firm (Guangzhou, China). For the FISH assay, RNA-FISH was carried out by utilizing a FISH Package (RiboBio, Guangzhou, China) in line with the producer’s protocols to evaluate the placement of circHIF1α in PIEC cells and PK-15 cells. Within the immunofluorescence assay, PK-15 cells have been transiently transfected with Cy3-labelled circHIF1α and proceeded to FISH assay, after which incubated with anti-IGF2BP3 antibody (Proteintech, Chicago, USA) to watch the colocalization of circHIF1α and IGF2BP3. All photographs have been noticed utilizing laser confocal microscopy (ZEISS, LSM800, Germany).
Twin-luciferase reporter assay
The plasmids (pmiRGLO-WT-HIF1α and pmiRGLO-MUT-HIF1α) have been synthesized and sequenced by Shandong Gene & Bio Co, Ltd (China). PK-15 cells have been transfected with plasmids. After 24 h of incubation, the cells have been analyzed with the Dualucif®Firefly & Renilla Assay Package (UElandy, Shanghai, China) in line with the producer’s directions. Detailed supply info for vectors is on the market in Desk S3.
Western blot evaluation
Complete proteins of exosomes and PK-15 cells have been extracted with RIPA lysis buffer containing PMSF. The protein focus was measured by the BCA technique. After being purified and electrophoretically separated, proteins have been transferred to PVDF membranes (Millipore, Billerica, USA). The membranes have been blocked with 5% skimmed milk for 1 h after which incubated in a single day at 4 °C with every major antibody and subsequently incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Lastly, the improved chemiluminescence system (BeyoECL Moon, Beyotime, Jiangsu, China) was utilized for sign visualization. Grey values of protein bands have been quantified by Picture J software program. All antibodies of Western blot used on this research have been listed in Desk S4.
Co-immunoprecipitation (CoIP)
PMSF was added in 400 µL lysate containing 3 mg complete protein, and 4 µg particular antibody was added on the similar time. Furthermore, an equal quantity of homotypic IgG antibodies was added to an equal quantity of protein lysates, which was the IgG management group. All samples have been combined in a single day at 4 °C. Then 50 µl suspended ProteinA agarose beads have been added and incubated at 4 °C for 4 h. The precipitated advanced was washed 5 instances with 1mL 1 × TBST added with PMSF. Lastly, the proteins have been detected by Western blot.
Micro organism adhesion and invasion assays
Adhesion and invasion assays have been carried out utilizing PK-15 cells. A complete of 5 × 105 PK-15 cells have been cultured in a single day at 37 °C with 5% CO2 after which contaminated with totally different micro organism to permit bacterial adhesion. On this research, PK-15 cells have been contaminated with S. aureus (MOI = 1) for 4 h, SS2 (MOI = 10) for 8 h, and G. parasuis (MOI = 10) for 12 h, respectively. Cells have been rigorously washed 5 instances with PBS to eradicate non-specific bacterial attachment after which incubated for 10 min at 37 °C with 100 µl 0.25% trypsin-EDTA. After incubation, 900 µl ice-cold PBS was added, and cells have been faraway from the tradition plates by scraping the underside of the nicely with a sterile scalpel blade. The cell suspensions with adherent micro organism have been diluted and put onto the associated plates containing NAD and serum. For the micro organism invasion assay, DMEM containing 100 µg/mL gentamicin was added to the PK-15 cells cultured, which have been washed twice with PBS. The cells have been additional incubated for 30 min to verify that extracellular micro organism have been killed. Monolayers have been washed thrice with PBS and intracellular micro organism have been harvested as above. All the above assays have been carried out in triplicate and replicated thrice.
NSC 80467 (MCE, New Jersey, USA) is a DNA damaging agent that induces markers of DNA injury γ-H2AX. Thymidine (MCE, New Jersey, USA) is a DNA synthesis inhibitor that arrest the cell on the G1/S boundary previous to DNA replication. Herein, NSC 80,467 or Thymidine was used to deal with the PK-15 cells after siRNAs concentrating on circHIF1α have been transfected into PK-15 cells, then PK-15 cells contaminated with totally different micro organism (S. aureus, SS2 or G. parasuis) and the variety of PK-15 cells adhered and invaded by micro organism in line with the above operation technique, respectively.
Animal problem
All animal experiments have been accepted by the Animal Experimental Moral Inspection Type of the Institute of Animal Science and Veterinary Medication, Shandong Academy of Agricultural Sciences (IASVM-2022-012). 6–8 weeks previous feminine BALB/c nude mice have been obtained from Jinan Pengyue Laboratory Animal Breeding CO., Ltd. (Shandong, China). With a view to decide the resistance of exosomes derived from PIEC cells to bacterial an infection in animals, PIEC cells have been contaminated with totally different micro organism (G. parasuis, SS2, and S. aureus) respectively. Then, the exosomes derived from PIEC cells contaminated with totally different micro organism have been remoted. The exosomes of various concentrations (10 mg/kg, 20 mg/kg, and 30 mg/kg) have been injected by way of the tail vein of mice (n = 6 for every group). The distribution of exosomes in mice at totally different instances after injection was noticed utilizing Bioluminescence imaging (Tanon, Shanghai, China).
To make clear the consequences of exosomes and circHIF1α on bacterial an infection, exosomes labeled with DiR (MedChemExpress, New Jersey, USA) have been injected in line with the strategy described above (n = 6 mice/group), and PK-15 cells with overexpression of circHIF1α or their empty vectors have been injected by way of the tail vein of mice (n = 6 mice/group). After 24 h, management mice and mice injected exosome or circHIF1α have been challenged with totally different micro organism (G. parasuis, SS2 or S. aureus) by intraperitoneal injection, respectively. The an infection dose of G. parasuis was 5 × 109 CFU, SS2 was 4 × 109 CFU, S. aureus was 7.5 × 109 CFU. On the similar time, the exosome management group (n = 6 mice/group) and circHIF1α management group (n = 6 mice/group) with out bacterial an infection have been established. The Scientific signs and mortality of mice have been constantly noticed for 14 d. The mice on the point of dying throughout the experiment and people nonetheless alive on the 14th d have been euthanized. Completely different organs have been fastened, embedded in paraffin, and sectioned. The sections have been used for hematoxylin and eosin (H&E) staining. The bacterial content material in several organs was decided by viable bacterial depend and real-time PCR.
Knowledge and statistical evaluation
Statistical analyses have been carried out utilizing GraphPad Prism 8.00 Software program (GraphPad Software program Inc., USA). Scholar’s t-test (two-tailed) was employed when evaluating between two teams. The Wilcoxon take a look at and one-way ANOVA have been used to match measurement knowledge amongst a number of teams. p < 0.05 was thought of statistically vital (*p < 0.05, **p < 0.01, ***p < 0.001).
