Effect Of PSMB5 On The Proliferation Of Neural Stem Cells

Objective:To explore the mechanism of neural stem cells(NSCs) proliferative potential is decreased with the increasing age by RNA inference and observed the effect of 20 S proteasome beta-5 subunit(PSMB5) on proliferation of NSCs,which would further provide new ideas for treating neurodegenerative diseases. Methods:1.Different aging subventricular zone(SVZ) NSCs of mice for cultruing in vitro, which are Embryonic 14 days(E14), Postnatal day(P0),Postnatal 30 days(P30),Postnatal 90 days(P90),Postnatal 540 days(P540). Different aging NSCs of mice for cultruing in vitro,the proliferation capacity in the different aging NSCs were observed by the formation rate of neurosphere. 2. Extract the E14, P0, P30, P90, P540 mouse SVZ protein and analysis the level of PSMB5 expression at different ages by western blot.3. Design and synthesize PSMB5-si RNA and PSMB5-si RNA-NC.The E14 NSCs were infected with PSMB5-si RNA or PSMB5- si RNA-NC by nuclefecrtor. The expression of PSMB5 m RNA and protein were analyzed by real-time PCR and western blot. The proteasome activity was detected by fluorescence spectrophotometry. 4.Brd U incorporation assay and the formation rate of neurospheres were used to reveal the effect of PSMB5 knockdown on NSCs proliferation. 5.The protein levels of Cyclin-D1 and CDK4 were detected by western blot. Results:1.The neurosphere forming cycle of P30,P90 and P540 mice is longer than E14 and P0 mice in vitro. Tips to increase with age the NSCs proliferation potential is decreased. At the same time, western blot results show that the expression of PSMB5 also gradually decreased with increasing age,which imply PSMB5 may be involved in the regulation of NSCs proliferation.2. After transfection for 48 hours and 72 hours, the m RNA and protein level of PSMB5 were decreased by 61.23±1.62% and 40.11±2.45% respectively(P<0.05), compared with control group, which showed that the expression of PSMB5 gene was significantly declined by si RNA.Moreover, the proteasome activity of NSCs was significantly reduced by 24.36±0.14% in si RNA group(P<0.01). 4.Following PSMB5 knockdown of NSCs,the percentage of Brd U-possitive cells lowered from 53.47±7.36% to 21.58±3.29%(P<0.05)and the formation rate of neurosphere was significantly lower than the control group. Furthermore, the protein levels of Cyclin D1 and CDK4 were substantially reduced by 46.82±3.14% and 32.86±2.75%(P<0.05)after PSMB5 gene silencing,which show that reduced cell proliferation in NSCs through down-regulation of the cell-cycle regulator Cyclin D1 and CDK4. Conclusions:1. PSMB5 might be involved in regulation NSCs proliferation.2.The proliferation capacity of the NSCs was inhibited through down-regulation of the cell-cycle regulator cyclin-D1, CDK4 and proteasome activity by PSMB5 knockdown, which suggested that PSMB5 may be a key target to the cell vitality of NSCs.