The Construction Of CIAPIN1 Differential Expression Model And Application In Rabies Virus Proliferation Study

There was a new confirmed anti-apoptotic molecule named CIAPIN1(cytokine-induced apoptosis inhibitor 1)recently and widely expressed in various tissues of the body.CIAPIN1 involved in hematopoiesis,anti-cancer drug resistance regulation,cancer development and anti-apoptotic process of cells.There were some studies showed that CIAPIN1 is distributed in central nervous tissues widely and can be used as an effector molecule in some neurodegenerative diseases.Rabies virus is a highly lethal neuroinvasive virus which can causes a variety neurological dysfunction disease and induces neuronal apoptosis.Our previous study showed that CIAPIN1 was expressed differently in brain when mouse was infected with rabies virus in different virulence.Therefore,we want to observe whether the expression of CIAPIN1 will affect proliferation of rabies virus and provide reference for the production of high-priced vaccines.Here,we constructed a CIAPIN1 overexpressing model named N2a-CIAPIN1-OVER and a CIAPIN1 silence model in N2 a cell line by CRISPR-Cas9 and RNA interference.Detect the CAIPIN1 expression of model with real-time PCR and Western Blotting.We detected proliferation ability of the model with CCK8,observed cell cycle and early apoptosis in flow cytometry and we also detected the m RNA expression of Caspase3,Bcl2,Bax and Caspase9 which are marker of apoptosis by real-time PCR.At last we infected model with rabies virus in differently virulence then detected the virus titer and expression of the viral N gene for ensure the change of virus proliferation.Our results are as follow.There are some different in CIAPIN1 expression between the control groups and experiment groups.Compare to control groups,over expression groups were(274±3.06)%(P<0.05)and silence groups were(35±2.18)%(P<0.05)in CIAPIN1 m RNA expression.After detected by Western Blotting,over expression groups were(210±1.94)%(P<0.05)compare to control groups and silence groups were(18±1.14)%(P<0.05).Our result showed that the model was constructed successfully.The cell proliferation and cell cycle influenced by the expression of CIAPIN1.Specifically,the cell proliferation activity of the over expression groups are stronger than the control groups(P<0.05)and more cells were in G2/M cycle.The proliferation activity of the silence groups was weaker than the control groups(P<0.05)and more cells were arrested in the S cycle.All indicated that the expression of CIAPIN1 can affect the proliferation by affecting the cell cycle.Compared with the control groups,the overexpression groups and the silence groups showed different in expression of apoptosis-related genes.The apoptotic factors caspase9,Bax,caspase3 were significantly up-regulated(P<0.05),while the anti-apoptotic factor Bcl-2 was significantly down-regulated(P<0.05),and the silence groups showed more obvious changes than over expression groups.It was demonstrated that altering the expression level of CIAPIN1 induces the change of the expression of apoptosis-related genes,which suggest the occurrence of apoptosis.The results of virus proliferation showed that the virus titers and viral N gene expression levels of different virulence viral in the over expression groups or in the silence groups were lower than those in the control group.Conclusion: our result showed that the effective change of CIAPIN1 expression will leads to proliferation change of rabies virus which is mainly affected by cell viability and apoptosis.Lay the foundation for the development of high-cost rabies vaccine...