Analysis of deep sequencing Exosome-microRNA expression profile from Chicken Type Ⅱ Pneumocytes derived reveals potential role of gga-miRNA-451 in inflammation
Exosomes are nanosized extracellular vesicles secreted by multiple cells in the body, including those located in the respiratory tract and lungs. They are emerging as important inflammatory mediators and can release their contents, especially microRNAs (miRNAs), to both neighboring and distal cells. Mycoplasma gallisepticum (MG) can target host cell and cause chronic respiratory disease (CRD) in chickens. Although exosomal miRNAs have been demonstrated to produce an important effect on
... effect on microbial pathogenesis and inflammatory response as crucial regulatory noncoding RNAs, the mechanism by which exosomal miRNAs regulate MG-induced inflammation remains to be elucidated. Methods: the expression of exosome-microRNA derived from MG-infected chicken type Ⅱ pneumocytes (CP-Ⅱ) was screened, and the target genes and function of differentially expressed miRNAs (DEGs) were predicted. To verify the inflammatory functions of exosomal gga-miR-451 via targeting YWHAZ, Western blot, ELISA, and RT-qPCR were used in this study. Results: A total of 722 miRNAs were identified from the two exosomal small RNA (sRNA) libraries, and 279 novel miRNAs were discovered; 30 miRNAs (9 up-regulated and 21 down-regulated) were significantly changed (P 0.05). Function annotation analysis of DEGs showed that the target miRNAs were significantly enriched in treatment group, such as cell cycle, Toll-like receptor signaling pathway and MAPK signaling pathway, etc. The results have also confirmed that gga-miR-451-absent exosomes derived from MG-infected CP-Ⅱ cells increased inflammatory cytokine production in DF-1 (chicken embryo fibroblast) cells, and Wild Type CP-Ⅱ cells-derived-exosomes displayed protective effects. Conclusion: our work suggests that exosomes from MG-infected CP-Ⅱ cells alter the dynamics of the DF-1 cells, and may contribute to pathology of the MG infection via exosomal gga-miR-451 targeting YWHAZ involving in inflammation. This could potentially be used as a biomarker for diagnostics and treatment. Background Mycoplasma galisepticum (MG) is one of the most important avian pathogens. It is established that (double-stranded chemically modified oligonucleotides) were synthesized by GenePharma (Shanghai, China). The sequences of all the primers and the sequences of RNA oligonucleotides used in the study are shown in Table S1 and Table S2 , respectively. CP-II Isolation and DF-1 Cells Culture CP-Ⅱ cells were isolated as described previously (24) , with modifications. Briefly, lungs were removed from 14-day-old of White Leghorn specific-pathogen-free (SPF) chicken embryos and were cut into very small tissue blocks. Then 0.25% trypsin and 0.1% IV collagenase (Invitrogen-Gibco, Carlsbad, CA, USA) were used for digestion at 37 ℃ for 10 min and 15 min, respectively. Cell suspensions were filtrated by a 200-mesh sieve, resuspended with 10 % fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) in a 150-mm sterile culture plate, and incubated for 1 h. Supernatants with the unattached cells were then collected three times. The unattached cells were centrifuged at 1200 r/min for 5 min, re-suspended in fresh Dulbecco's modified Eagle's medium (DMEM) for three times, and filtrated by 400 mesh sieves. After the cell count was completed, cells were with 20% FBS and concentration was adjusted to 2×10 6 /ml and then inoculated into 150-mm sterile culture plate. Cells were incubated for 18 h at 39 °C. The attaching cells on culture dish were CP-Ⅱ cells. Invert microscope and transmission electron microscope (TEM) were used to identify the cells. The chicken embryonic fibroblast cell line (DF-1), obtained from ATCC (Manassas, VA, USA), were maintained at 39 °C in a 5% CO 2 atmosphere in DMEM (Invitrogen, Carlsbad, CA, USA) mixed with 1% penicillin-streptavidin-glutamine (PSG, Invitrogen, USA) and 10 % fetal bovine serum (FBS, Invitrogen, USA). 2.3 Mycoplasma strains MG-HS, a virulent strain, was isolated from a chicken farm in Hubei, China and conserved in the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University (Wuhan, China) (25, 26 ). The MG-HS strain was cultured, and the concentration was determined as previously described.