Study On The Interaction Of The NF-κB Subunits And The Tumor Suppressor Gene Maspin

Objective To explore the interaction between the NF-κB family members and the tumor suppressor Maspin, which may further interferes with the biological behaviors of prostate cancer cells. Methods The expression of NF-κB subunits and Maspin was detected and performed to detect the expression of RelA and RelB with proteasome inhibitor by Western blot analyses in prostate cancer cells. RNA interference was performed to analyze how RelB-or RelA-deletion affected cell death as well as the expression of NF-κB subunits and Maspin; the regulation of RelB on Maspin expression in prostate cancer cells was also studied via stable transfection of RelB expression plasmid. Specific shRNA targeting the maspin gene was designed. The plasmid targeting the maspin gene was constructed and lentiviral expression system was used for transfection. qRT-PCR and Western blot were performed to identify maspin-shRNA stably transfected PC-3cells. qRT-PCR was used for analyzing the apoptosis-related genes. The xCELLigence system was applied for dynamic observing cell growth and calculating doubling time. Flow cytometry analysis was performed to detect cell death upon treated with proteasome inhibitor. Results RelA, p50, RelB, and p52were constitutively expressed in androgen-independent prostate cancer cell lines DU145and PC-3, while RelB had the highest expression in DU145cells. RelB-deletion in DU145cells by siRNA interference upregulated the endogenous expression of Maspin and induced cell death. But RelA-deletion had no significant influence on the endogenous expression of Maspin. Overexpression of RelB in PC-3cells inhibited the endogenous expression of Maspin. The recombinant plasmid containing maspin-shRNA was successfully constructed. Limited dilution was performed to obtain monoclonal PC-3-siMaspin cells. Cell growth of PC-3-siMaspin was significantly faster than that of PC-3-control cells (p=0.03). Doubling time of PC-3-siMaspin was26.83h while that of PC-3-control cells37.95h. The mRNA expression of bcl-2and A20in PC-3-siMaspin cells was increased; while that of box and bim was down. The cell death frequency of PC-3-control and PC-3-siMaspin cells8h after treated with MG-132was27.1%±5.6%and7.47%±2.3%,24.2%±3.7%and8.2%±2.5%at24h,28.7%±3.7%and7.6±2.5%at36h. RelA expression was decreased in PC-3-control cells upon treated with MG-132while that of PC-3-siMaspin cells stayed unchanged. Conclusion The classical and alternative NF-κB activities were sustained in androgen-independent prostate cancer cell lines. The expression of RelB and Maspin were inversely correlated in androgen-independent prostate cancer cells. The expression of RelB negatively regulated the endogenous expression of Maspin, which interfered with cell death. RelA was not involved in the regulation of Maspin expression. Maspin expression was increased in the androgen-independent PC-3prostate cancer cells. Maspin-depletion significantly reduced the doubling time and accelerated cell growth. Maspin silencing markedly reduced sensitivity of PC-3cells to proteasome inhibitor, which was linked to the abolishment of RelA reduction.