| Reticuloendotheliosis(RE) is an immunosuppressive avian tumor disease caused by reticuloendotheliosis virus(REV) infection.It is widespread in domestic and foreign aquaculture industries,which has caused large losses.Because the current research on this disease is relatively limited,especially its tumorigenesis mechanism,in order to effectively reduce the harm of RE to the breeding industry,it is urgent to understand the detailed mechanism of its incidence.As a recognition substrate of ubiquitin ligase complex(SCF),S-phase kinase-associated protein 2(Skp2)is closely related to the abnormal proliferation of cells and has attracted widespread attention in tumor research.After looking through the related research about the Skp2 gene in the world,as one of the current research hotspots in the medical,biological,and especially in veterinary medicine fields,it has been mainly studied in mammals,especially in human diseases.And about the relationship between the avian Skp2 gene and the cell cycle abnormalities caused by REV has not been reported so far.In this study,short hairpin RNA(sh RNA)-mediated Skp2 silencing plasmid vectors were successfully constructed and transfected into REV-infected SPF chicken embryo fibroblasts(CEFs).Furthermore,RT-PCR and Western Blot were used to systematically study the changes of Skp2,p27,Cyclin D1 mRNA expression and protein content at the transcription and protein levels after SPF CEFs infection with REV,respectively.Flow cytometry combined with PI-single-staining method was used to detect the cell cycle changes of SPF CEFs infected with REV.The CCK-8 Kit was used to conduct a comprehensive and systematic study on the proliferation of REV-infected SPF CEFs.This study helps to further understand the mechanism of cell cycle abnormalities caused by REV and find new molecular targets for the prevention of RE.The main results are as follows:(1)In this study,the chicken Skp2 gene real-time fluorescent quantitative PCR detection method was successfully established.The optimal reaction system of this method is 20.0 ?L: including Real-time PCR Master mix(2×)10.0 ?L,and the upstream and downstream primers(10 ?mol/L)1.0 ?L,c DNA 2.0 ?L,dd H2 O 6.0 ?L;optimal reaction conditions are pre-denaturation at 95? for 10 s,denaturation at 95? for 5 s,annealing/extension at 60? for 34 s,30 cycles.(2)The expression of Skp2 mRNA at 6h,12 h,24h,48 h,72h,96 h and 120 h after SPF CEFs infection with REV was higher than that of the uninfected control group,especially at 24 h,48h and 72 h,the increase of Skp2 mRNA expression were extremely significant(P<0.001).(3)Compared with uninfected control group,the proliferation vitality of CEFs significantly increased(P<0.05 or P<0.01)at 12 h,24h and 48 h after REV infection with SPF CEFs.Compared with REV-infected CEFs group,the proliferation vitality of CEFs cells after transfection with sh RNA-358 and sh RNA-141 were significantly decreased(P<0.01);there was no statistically significant difference in proliferation activity of CEFs in V-sh RNA-NC group(P>0.05).(4)In the four successfully constructed Skp2 expression suppression plasmids,sh RNA-908,sh RNA-358,sh RNA-225 and sh RNA-141,the inhibitory effect of sh RNA-358 and sh RNA-141 on Skp2 were extremely significant increased(P <0.001)at 24 h,48h and 72 h after transfection.(5)Compared with the blank control group,the expression of p27 mRNA in SPF CEFs infected with REV was significantly reduced(P<0.001);the expression of Cyclin D1 mRNA was significantly increased(P<0.001).Compared with the REV-infected CEFs group,the expression levels of Skp2 and Cyclin D1 mRNA decreased significantly(P<0.001 or P<0.01),while change of p27 mRNA expression was not significant(P> 0.05).After the plasmid sh RNA-NC acted on CEFs,the mRNA expression levels of the mentioned indicators above were not statistically different(P>0.05).(6)Compared with the blank control group,the expression of Skp2 and Cyclin D1 protein in REV infected SPF CEFs were significantly increased(P<0.01),and the expression of p27 protein was significantly reduced(P<0.001).Compared with REV-infected CEFs group,the expression levels of Skp2 and Cyclin D1 protein in CEFs transfected with sh RNA-358 and sh RNA-141 were significantly reduced(P<0.01 or P<0.001);the expression of p27 protein was significantly increased(P<0.05).After the plasmid sh RNA-NC acted on CEFs,the protein expression levels of the tested indicators above had no statistical changes(P>0.05).(7)After CEFs were infected with REV,the number of cells in G0/G1 phase was significantly reduced(P<0.01),and the number of cells in G2/M phase was significantly increased(P<0.01),cell numbers in S phase had no statistical difference(P>0.05);compared with the REV infection group,the number of cells in the G0/G1 phase of the V-sh RNA-358 group and the V-sh RNA-141 group were significantly higher than that of the control group(P<0.01),and the number of cells in the G2/M phase was significantly(P<0.01)decreased,cell numbers in S phase had no statistical difference(P>0.05);moreover the cell number of each phase between the V-sh RNA-NC group and REV infection group had no significant change(P>0.05).The results above provide an important scientific experimental basis for further clarifying the effect of REV infection on the proliferation and cycle of CEFs and the role of Skp2 during the infection.It also provides new ideas for further exploring the immunopathogenic mechanism of REV at the level of molecular regulation. |
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