Active Constituent Of Polyporus Induces Apoptosis And Inhibits Proliferation Mediated By HuR In T24 Cell

Objective:Polyporus is medicinal fungus, with functions of enhancing immunity, antitumoi which has obtained the good curative effect such as clinical application in the treatment o bladder cancer, however the mechanism has not been fully elucidated. The main chemica composition of polyporus include polysaccharides and nonsteroidal, another protein, biotin etc. This experiment mainly study the mechanism of active constituent of Polyporu: induces apoptosis and inhibits proliferation Mediated by HuR in T24 Cell. Through the experiment to achieve the aims as follows:1. Research the effect of HuR on the expression of BCL-2 mRN A in T24 cells.2. Research the effect of PPS on the HuR-mediated expression of BCL-2 in T24 cells.3. Research the effect of ergosterol on the HuR-mediated expression of cyclin D1 in T24 cells.Methods:1. Research the effect of over-expressed HuR on the expression of BCL-2 mRN A in T24 cells.T24 cells were cultured in antibiotics-free medium for 24 h before transfection. Cells were divided into three groups:control group (without treatment), group transfected with empty vector and group transfected with vector containing HuR coding sequence. The expression level of HuR was determined by western blotting.The effect of over-expressed HuR on the stability of bcl mRNA in T24 cells was analyzed by follows:Actinomycin D was added to inhibit RNA systhesis, cells were collected to pick out RNA at 0 h,2 h,4 h and 6 h, followed by RT-PCR to determine the half-life of BCL-2 RNA.The effects of over-expressed HuR on the expression level of bcl mRNA and BCL-2 protein were analyzed by RT-PCR or western blotting.2. Research the effect of silenced HuR on the expression of BCL-2 in T24 cells.SiHuR was used to transfect T24 cells. Cells were divided into three groups:control group (without treatment), negative control group (transfected with negative control siRNA) and treatment group (transfected with siRNA targeting HuR coding sequence).24 h post-transfection, actinomycin D was added to inhibit RNA synthesis and then the levels of BCL-2 mRNA were determined and the half-life of BCL-2 mRNA was calculated. The effect of silenced HuR on the stability of BCL-2 mRNA was then analyzed.HuR gene silencing vector was constructed and RT-PCR was used to detect the effects of silenced HuR on the expression level of bcl mRNA and BCL-2 protein were analyzed by RT-PCR or western blotting.3.The effect of PPS on the HuR-mediated expression of BCL-2 in T24 cells.T24 cells were divided into four groups:control group (without treatment), PPS-low dose group (50 μ g/mL PPS), PPS-moderate dose group (100 μ g/mL PPS)and PPS-high dose group (200 μ g/mL PPS).Before pharmacological experiment, MTT method was used to draw cell growth curve to know the growth status of T24 cells and determine rational cell seeding density. The effect of PPS on the apoptosis of T24 cells was detected by Hoechst staining and flow cytometry, respectively.Research the effects of PPS on the expression of BCL-2 in T24 cells:Western blotting and RT-PCR were used to detect the effects of PPS on the expression of BCL-2 and BCL-2 mRNA at different concentrations.Research the effect of PPS on the BCL-2 mRNA in T24 cells:Relative expression level of BCL-2 mRNA in each group was determined by RT-PCR after addition of actinomycin D. The mRNA level at 0 h was set as 100% to calculate the relative levels of mRNA at 2 h,4 h and 6 h so as to determine the half-life of BCL-2 mRNA. Finally, the effect of PPS on the stability of BCL-2 mRNA was analyzed.Research the effect of PPS on the distribution of HuR in T24 cells:Cells in each group were treated with PPS at different concentrations for 24 h and then were collected and fractionated to extract cytoplasmic and nuclear proteins for western blotting analysis to locate the distribution of HuR.The effect of PPS on the content of BCL-2 mRNA in HuR-mRNP complex:Cells were lyzed to prepare lysate containing mRNP and then nonspecific antigens were removed. mRNP complex was immunoprecipitated with antibody-conjugated agarose beads and then total mRNA was purified and target gene was amplified to determine the effect of PPS on the binding of BCL-2 mRNA with HuR in T24 cells.4. The effect of ergosterol on the HuR-mediated expression of cyclin D1 in T24 cells.T24 cells were divided into three groups:control group (without treatment),0.5μM ergosterol group,1 μ M ergosterol groupThe effect of ergosterol on the proliferation of T24 cells was detected by MTT and flow cytometry, respectively.The effects of ergosterol on the expression of cyclin Dl in T24 cells:Western blotting and RT-PCR were used to detect the effects of ergosterol on the expression of cyclin Dl and cyclin D1 mRNA at different concentrations.The effect of ergosterol on the cyclin D1 mRNA in T24 cells:Relative expression level of cyclin D1 mRNA in each group was determined by RT-PCR after addition of actinomycin D. The mRNA level at 0 h was set as 100% to calculate the relative levels of mRNA at 2 h,4 h and 6 h so as to determine the half-life of cyclin D1 mRNA. Finally, the effect of ergosterol on the stability of cyclin D1 mRNA was analyzed.The effect of ergosterol on the distribution of HuR in T24 cells:Cells in each group were treated with ergosterol at different concentrations for 48 h and then were collected and fractionated to extract cytoplasmic and nuclear proteins for western blotting analysis to locate the distribution of HuR.The effect of ergosterol on the content of cyclin D1 mRNA in HuR-mRNP complex: Cells were lyzed to prepare lysate containing mRNP and then nonspecific antigens were removed. mRNP complex was immunoprecipitated with antibody-conjugated agarose beads and then total mRNA was purified and target gene was amplified to determine the effect of ergosterol on the binding of cyclin D1 mRNA with HuR in T24 cells. Results:1. The effect of over-expressed HuR on the expression of BCL-2 in T24 cellsOverexpression of HuR in control group and negative control group had no significant effects on the degradation of BCL-2 mRNA and expression levels of BCL-2 mRNA and BCL-2 protein. In comparison with control and negative control groups, degradation of BCL-2 mRNA in HuR overexpression group was significantly reduced and expression levels of BCL-2 mRNA and BCL-2 protein were significantly increased (p<0.05).2. The effect of silenced HuR on the expression of BCL-2 in T24 cellssilencing of HuR in control group and negative control group had no significant effects on the degradation of BCL-2 mRNA and expression levels of BCL-2 mRNA and BCL-2 protein. In comparison with control and negative control groups, degradation of BCL-2 mRNA in HuR silencing group was significantly increased and expression levels of BCL-2 mRNA and BCL-2 protein were significantly reduced (p<0.05).3. The effect of PPS on the HuR-mediated expression of BCL-2 in T24 cells.(1)The effect of PPS on the apoptosis of T24 cells:After treatment for 24 h, cell nuclei in the control group was evenly stained with Hoechst 33342 and no significant difference was observed between control group and PPS-low dose group. Cells in PPS-moderate dose and high dose groups showed morphology of apoptotic cells: chromatin condensation and hyperchromatic nuclei,T24 cells treated with PPS at different concentrations for 24 h were then double-stained with Annexin V-FITC/PI and detected with flow cytometry. Compared with control group, cell apoptosis in PPS-low dose group showed no significant difference while early phase apoptotic rates in PPS-moderate and high dose groups were significantly increased (p<0.05).(2) Comparison of expression level of BCL-2:Compared with control group, BCL-2 expression level in T24 cells treated with low dose PPS for 24 h showed no significant difference while were significantly increased in moderate and high dose PPS groups (p<0.05).(3) Comparison of expression level of BCL-2 mRNA:compared with control group by RT-PCR, expression level of BCL-2 mRNA in T24 cells treated with low dose PPS for 24 h showed no significant difference while it was significantly reduced in moderate and high dose PPS treatment groups (p<0.05).(4)Comparison of mRNA stability among groups:mRNA synthesis was inhibited by addition of actinomycin D after treatment for 24 h with PPS at different concentrations and its concentration was determined at 0 h,2 h,4 h and 6 h to draw mRNA degradation curve. In comparison with control group, the stability of BCL-2 mRNA in control group and PPS-low dose group showed no significant difference while it was significantly decreased in PPS-moderate and high dose groups(p<0.05).(5) Comparison of total, cytoplasmic and nuclear HuR:Total HuR protein in each group after treatment for 24 h with PPS showed no significant difference. In comparison with control group, cytoplasmic HuR in PPS-low dose group showed no significant difference and significantly reduced in PPS-moderate and high dose groups(p<0.05). In comparison with control group, nuclear HuR protein in PPS-low dose group showed no significant difference and was significantly increased in PPS-moderate and high dose groups(p<0.05).(6) Effects of PPS treatment for 24 h on the binding of BCL-2 mRNA and HuR:In comparison with control group, the amount of BCL-2 mRNA bound to HuR in PPS-low dose group treated with PPS for 24 h showed no significant difference and was significantly reduced in PPS-moderate and high dose groups(p<0.05).4.The effect of ergosterol on the HuR-mediated expression of cyclin D1 in T24 cells.Compared with the control, the cell growth were obviously inhibited (P<0.05), the cyclin D1 mRNA stability and cyclin D1 mRNA were decreased (P<0.05), the total protein of HuR was slightly changed, but the cytoplasm protein of HuR was down-regulated (P<0.05) and the nucleus protein of HuR was up-regulated (P<0.05). the amount of cyclin D1 mRNA bound to HuR was significantly reduced (P<0.05).Conclusion:Polyporu play the role of resistance to bladder cancer through PPS and ergosterol which its active constituent. PPS can promote T24 bladder cancer cell apoptosis, this effect may be mediated by change the expression of BCL-2 gene through HuR mediated post-transcription regulation in T24 cells; Ergosterol inhibits T24 cell proliferation, the effect may be mediated by change the expression of cyclin D1 gene through HuR mediated post-transcription regulation in T24 cells.