The Research About Mechanism Of The MTOR Signaling Pathway Taking Part In The Coxsackie Virus B3-induced Apoptosis Of HeLa Cells

Objective:In order to investigate the role of PI3K/Akt/mTOR signaling pathway playing in the CVB3induced cell apoptosis,we observed the change of CVB3indeuced apoptosis, cytopathic effect and the expression of pro-apoptotic factors via using the inhibitors of mTOR and PI3K respectively and gene transfection to build HeLa cells model with CVB3infected and together with inhibition of mTOR or PI3K signaling pathway and overexpression of their downstream factors.Methods:The HeLa cells were chosen to build the classic CVB3infected cell model. After intervention by Rapamycin-the inhibitor of mTOR and LY294002--the specific inhibitor of PI3K, apoptosis was determined by fluorescenceactivated cell sorting (FACS) analysis cells stained with Annexin-V-FITC and propidium iodide (PI) by the flow cytometer and the change of viral replication and the expression of the pro-apoptotic factors Bim, Bax, Caspase-9, Caspase-3were determined by semiquantitative PCR and Western bloting methods.The overexpression cell models of4EBP1, p70S6K, Aktl or Akt2were built through the gene transfection. After infected with CVB3,apoptosis was determined by fluorescenceactivated cell sorting (FACS) analysis cells stained with Annexin-V-FITC and propidium iodide (PI) by the flow cytometer and the change of viral replication and the expression of the pro-apoptotic factors Bim, Bax, Caspase-9, Caspase-3were determined by real-time fluorescence quantitative PCR, Western bloting methods, immunofluorescence and transmission electron microscope.Results:(1)Both Rapamycin and LY294002inhibit cell viability in a concentration-dependent manner.(2) Rapamycin and LY294002have different roles of CVB3replication but promote CVB3-induced CPE and apoptosis. (3) Rapamycin and LY294002block the ability of CVB3to prevent Bim activation wihle stimulate the CVB3-induced Bax activation.(4) Rapamycin and LY294002stimulate CVB3-induced procaspase-9activation and caspase-3self-cleavage to initiate the caspase cascade(5) After building the cell line overexpressed with empty vector, myc expression was determined by Western bloting which was found only in the empty vector group.(6) After building the cell lines overexpressed with pcDNA3.1-myc-HisA(-)-4EBP1, pcDNA3.1-myc-HisA(-)-p70S6K, pcDNA3.1-myc-HisA(-)-Aktl and pcDNA3.1-myc-HisA(-)-Akt2, respectively, the protein expressions were determined by Western bloting. The expression of4EBP1, p70S6k, Aktl or Akt2was much higher than controls in the corresponding group.(7) Overexpression of4EBP1, p70S6K, Aktl or Akt2promotes the CVB3-induced apoptosis and CPE.(8) Overexpression of p70S6K, Aktl or Akt2promotes viral mRNA expression but blocks the VP1expression, which is opposite to the4EBP1-transfected groups.(9) CVB3-induced Bim and Bax activation at the early stage are blocked by the overexpression of4EBP1, p70S6K or Akt2while promoted by overexpression of Aktl.(10) Overexpression of4EBP1or p70S6k provokes the CVB3-induced procaspase-9and caspase-3self-cleavage, which are blocked by the overexpression of Aktl or Akt2.Conclusion:(1)The inhibition of mTOR and PI3K has different regulatory pathways in the CVB3-induced apoptosis.LY294002promotes the apoptosis via upregulating the CVB3mRNA expression and Bim, Bax, caspase-9, caspase-3protein activation, while different from LY294002, rapamycin upregulates the viral protein synthesis instead of mRNA. Looking at everything as a whole, our study demonstrates that the PI3K and mTOR signal pathways participate in the CVB3-induced apoptosis through mediating the proapoptotic factors in Bcl-2and caspase families. (2) Eukaryotic expression vectors pcDNA3.1-myc-HisA(-), pcDNA3.1-myc-HisA(-)-4EBP1, pcDNA3.1-myc-HisA(-)-p70S6K, pcDNA3.1-myc-HisA(-)-Aktl and pcDNA3.1-myc-HisA(-)-Akt2are successfully built.(3) HeLa cells are successfully transfected with each Eukaryotic expression vector via application of liposomes and stable cell lines overexpressing4EBP1,p70S6K, Aktl and Akt2are established.(4) Overexpression of4EBP1,p70S6k, Aktl or Akt2promotes the CVB3-induced apoptosis through different mediation mechanisms. Overexpression of4EBP1or p70S6K promotes the CVB3-induced apoptosis via a Bax and Bim-independent pathway, and overexpression of4EBP1could stimulate the viral replication at the meantime.Differently, overexpression of Aktl or Akt2,promoting the CVB3-induced apoptosis through a caspase-independent pathway, facilitates CVB3replication at the early stage after infection and inhibits that due to the negative feedback inhibition of Akt phosphorylation.