| Chronic Respiratory Disease(CRD)is mainly induced by Mycoplasma gallisepticum(MG)with extremely high infection rate.CRD exposes the sufferer to secondary infections caused by other pathogenic microorganisms,causing huge economic losses to the world’s poultry industry.It binds to specific receptors and enters the blood circulation through the host’s respiratory mucosal barrier,resulting in infection of organs and tissues throughout the body after MG infection.While,its pathogenic mechanism has not yet been elucidated.It has been confirmed that exosomes can regulate pathogenic microorganism infection by carrying miRNAs with the circulatory system to enter adjacent and distant cells or tissues.Therefore,exploring the role of exosomal-miRNAs in MG infection have great values.The previous high-throughput sequencing has found that in MG-Exo group,exosomes isolated from MG-HS infected chicken the primary alveolar type Ⅱ epithelial cells(CP-Ⅱs),ggamiR-193 a was significantly increased.In this study,exosomal-gga-miR-193 a is selected as the research object,CP-Ⅱs works as donor cells,the chicken embryo fibroblast cell lines(DF-1)functions as recipient cells,The aim of this study is to explore the function and mechanism of exosomal-gga-miR-193 a in MG infection.Results as follows:1.Ultracentrifugation and density gradient centrifugation were used to isolated exosomes.Nanoparticle Tracking Analysis was(NTA)was used to detect the size of exosomes,and the size was 30-150 nm.Transmission Electron Microscopy(TEM)was observed the shape of exosomes small and double membranes vesicles.Western blot was detected the marker proteins CD63 and CD9.The results of q PCR show,the relative expression of gga-miR-193a-3p in MG-HS infection exosomes,CP-Ⅱs and DF-1 cells were significantly increased(p<0.01)compared with uninfected groups(p<0.05).2.Fluorescent labeling technology was used to label the exosomes.PKH67 labeled the exosomes secreted by CP-Ⅱs,Cy3 labeled gga-miR-193a-3p.Exosomes were cocultured with the recipient cells for 8h,and found that exosomes can carry gga-miR-193 a to DF-1 cells.The results of q PCR have showed that the relative expression of gga-miR-193a-3p in the MG-HS-infected group significantly increased(p<0.01)compared with the normal group.gga-miR-193a-3p was over-expressed in CP-Ⅱs,exosomes were extracted from CP-Ⅱs.Total volume of 10μL,100μL and 500μL exosomes were incubated with the recipient cells respectively,and the cells were collected after 24 h incubation.Compared with the negative control,the relative expression of gga-miR-193a-3p in the recipient cells were significantly up-regulated after the addition of 100 μL and 500 μL exosomes(p <0.01).The results suggest that exosomes can carry gga-miR-193a-3p into the recipient cells,and it shows exosomes does-dependent manner.3.To study the effects of exosomes highly expressed gga-miR-193a-3p on the proliferation and apoptosis of recipient cells.DF-1 cells were incubated with MG-HS infection exosomes(MG-Exo)and normal group exosomes(Nor-Exo),and the other groups were transfected with gga-miR-193a-3p mimics and miR-NC.The results of CCK-8 have showed that the number of cells in the MG-Exo group was significantly downregulated than Nor-Exo group(p<0.01)after 24 hours,and there was no significant difference between the gga-miR-193a-3p mimics group and the miR-NC group;After 36 hours,the number of cells in the MG-Exo group was significantly downregulated than NorExo group(p<0.01),the number of cells in the gga-miR-193a-3p mimics group was significantly downregulated than miR-NC group(p<0.05),the number of cells in MG-Exo group was significantly upregulated than miR-NC group(p<0.01);After 48 hours,the results of flow cytometry have showed that the apoptosis rate in MG-Exo group was significantly upregulated than Nor-Exo group(p<0.05).Meanwhile,the apoptosis rate of the gga-miR-193a-3p mimics group was significantly upregulated than miR-NC group(p<0.01),and the apoptosis rate of the MG-Exo group was significantly upregulated than that of the miR-NC group(p<0.01).That indicates that exosomes with highly expressed in gga-miR-193 a,inhibits the proliferation of receptor cells and promotes the apoptosis of receptor cells.4.To explore the effect of exosome highly expressed gga-miR-193a-3p on the inflammatory response of recipient cells.The group was divided as above.After 48 h,the results of ELISA have showed that the expression levels of inflammatory factors TNF-αand IL-1β in MG-Exo group were significantly downregulated than Nor-Exo group(p<0.01).The expression levels of inflammatory factors TNF-α and IL-1β in cells of ggamiR-193a-3p mimics group were significantly downregulated than miR-NC group and Nor-Exo group(p<0.01).That indicates that exosomes with highly expressed in gga-miR-193 a,inhibiting inflammatory response by uptake of recipient cells.5.miRDB,Target Scan and David were used to predicted the target gene of gga-miR-193 a,we found that KRAS was target gene of gga-miR-193 a.The results of dual luciferase reporter system proved that overexpression of gga-miR-193a-3p could significantly inhibited(p<0.01)the luciferase activity of the KRAS 3’UTR dual luciferase reporter gene,inhibition of gga-miR-193a-3p can significantly enhance(p<0.01)the luciferase activity of the KRAS 3’UTR dual luciferase reporter gene,but the luciferase activity of P-KRAS 3’UTR has no effect;The relative expression of KRAS in m RNA and protein levels can be significantly inhibited(p<0.01)by gga-miR-193 a mimics.However,the relative expression of KRAS in m RNA and protein levels can be significantly enhanced(p<0.01)by gga-miR-193 a inhibitor.It was confirmed that KRAS was the direct target gene of gga-miR-193a-3p,and gga-miR-193a-3p negatively regulates the target gene KRAS.6.To further confirm the direct target gene of gga-miR-193a-3p.We designed a rescue test,one group of DF-1 was transfected with overexpressed KRAS vectors pc DNA3.1-KRAS(KRAS group)and pc DNA3.1,then infected with MG-HS(KRAS-MG group and pc DNA3.1-MG group),another group of DF-1 cells were transfected with pc DNA3.1-KRAS first,and co-transfect with gga-miR-193a-3p mimics(KRAS-miR-193a).The relative expression of KRAS were detected after 48 h.The results of q PCR and Western blot have showed that the relative expression of KRAS in KRAS group was significantly upregulated than other groups(p<0.01),and the relative expression of KRAS in the pc DNA3.1-MG group was significantly downregulated than pc DNA3.1 group(p<0.01).It was further confirmed that KRAS was the direct target gene of gga-miR-193a-3p in MG-HS infection.7.To study the involvement of KRAS in the regulation of MG-HS infection by the RAS/ERK signaling pathway.DF-1 cells were transfected with pc DNA3.1-KRAS(KRAS group),pc DNA3.1,si-KRAS,and si-NC respectively.After 48 hours of transfection,q PCR results have showed that the relative expression of key genes BRAF,MAP2K1 and MAPK1 in RAF-MEK-ERK signaling pathway axis were significantly upregulated than pc DNA3.1group,and the interference groups got opposite results(p<0.01).Western blot results have showed that the phosphorylated protein(p-ERK)of ERK in the KRAS group was significantly upregulated than pc DNA3.1 group,and the interference result was opposite(p<0.01).It proves that KRAS may play a role by regulating the RAS/ERK signaling pathway.8.To confirm that KRAS directly plays a role in MG-HS infection by RAS/ERK signaling pathway.One group of DF-1 cells were treated with U0126 and DMSO respectively.The other group of DF-1 cells were transfected with pc DNA3.1-KRAS(KRAS group)and pc DNA3.1 respectively,and treated with U0126 in KRAS group(KRAS-U0126),transfected 48 h.The q PCR results have showed that the relative expression of ERK in U0126 group was significantly downregulated than DMSO group(p<0.01),and the relative expression of ERK in KRAS overexpression group was significantly upregulated than KRAS-U0126 group and pc DNA3.1 group(p<0.01).It was confirmed that KRAS directly played a role in MG-HS infection through the RSA/ERK signaling pathway.9.To study the effect of gga-miR-193a-3p targeting KRAS on the proliferation and apoptosis of recipient cells.CCK-8 and flow cytometry were used to detect the cell proliferation and apoptosis of DF-1 cells.DF-1 cells were transfected with recombinant plasmids pc DNA3.1-KRAS(KRAS group),si-KRAS,U0126,pc DNA3.1,si-NC and DMSO.The results of CCK-8 have showed that at 24 h and 36 h,the number of cells in the KRAS group was significantly higher than pc DNA3.1 group(p<0.05);the number of cells in the si-KRAS group and U0126 group was significantly lower than si-NC group and DMSO group(p<0.01).The results of flow cytometry have showed that the apoptosis rate in KRAS group was significantly upregulated than pc DNA3.1 group(p<0.05).In addition,the apoptosis rate in the si-KRAS group and U0126 group were significantly lower(p<0.01)si-NC group and the DMSO group.It’s confirmed that the highly expressed gga-miR-193a-3p of exosomes inhibits the apoptosis and promote the proliferation of the recipient cells by targeting KRAS to activate the RAS/ERK signaling pathway.10.To explore the effects of KRAS and signaling pathway RAS/ERK on the inflammatory response of recipient cells.DF-1 cells were transfected with recombinant plasmids pc DNA3.1-KRAS(KRAS group),si-KRAS,U0126,pc DNA3.1,si-NC and DMSO.The results of ELISA detection show that the relative expression levels of proinflammatory factors TNF-α and IL-1β in KRAS group were significantly upregulated than pc DNA3.1 group(p<0.01);However,the relative expression of pro-inflammatory factors TNF-α and IL-1β in si-KRAS and U0126 groups were significantly downregulated than siNC group and DMSO group(p<0.01).Based on the above results,this study clarified that gga-miR-193a-3p,which was highly expressed in exosomes of MG-HS infected cells,resisted inflammatory response by target KRAS to activate the RAS/ERK signaling pathway against MG-HS infection in uninfected cells. |
