Effect Of Atg7 On The Proliferation And Differentiation Of Neural Stem Cells Of The Mouse

Objective:To investigate the key targets of NSCs proliferative and differential potential,we observe the influence of Atg7 on the potential of proliferation and differentiation of neural stem cells(NSCs)by RNA inference,which would offer new feasible strategy for treatment of neurodegenerative diseases.Methods:1.Design and synthesize Atg7-siRNA and control-si RNA.The P0 NSCs were infected with Atg7-siRNA or control-siRNA by nuclefecrtor.After transfection for 48 h and 72 h,total RNA and protein of NSCs were extracted.The expression of Atg7 mRNA and protein were analyzed by real-time PCR and Western blot.2.After knockdown Atg7,to detect the ability of autophagy of NSCs,we observed the expression level of LC3 II and P62 protein.3.The formation of neurospheres and BrdU incorporation assay were used to reveal the effect of Atg7 knockdown on the ability of proliferation of NSCs.4.Tuj1 and GFAP immunofluorescence staining were performed to analyse the influence of the potential of differentiation of NSCs.5.The application of senescence associated-β-galactosidase(SA-β-gal)staining identified senescent cells of NSCs after knockdown Atg7 and through the qPCR to detect the transcription of aging related gene P16,P21,P27 and P53.Results:1.After transfection for 48 hours and 72 hours,the real-time PCR showed that theexpression of Atg7 mRNA for NSCs in the siRNA group were significantly reduced almost 54%(P<0.001),compared to the control-siRNA group.And at the same time after knockdown Atg7,autophagy related genes such as Atg3,Atg5,Beclin1 mRNA level had no statistical significance.Western blot showed that the level of Atg7 in siRNA group was35% lower than the control group significantly(P < 0.01).2.The result of Western blot found that in siRNA group the expression of LC3 II protein was reduced by 27.5%(P <0.05),and the level of P62 protein were raised almost 1.5 times,compared to the control group(P < 0.001).3.The number of neurospheres were significantly declined 20%(P <0.01)and average diameter of neurospheres were lower 30%(P < 0.01)than control group,the result of BrdU incorporation showed the positive rate of BrdU decreased 29.8%compared with the control group significantly(P < 0.001).4.Tuj1 and GFAP Immunofluorescence staining showed,si RNA experimental group the positive rate of Tuj1 was 38.3% lower than the control group significantly(P < 0.05),while the positive cells rate of GFAP raised about 1.8 times than the control group(P < 0.01).Thus,those results showed that prompt Atg7 gene silencing can inhibit the ability of proliferation of NSCs and can also suppress the ability of differentiation to the direction of neurons.5.SA-β-gal dyeing were used to detect senescence cells,compared to the control group,Atg7-si RNA group raised about 2.8 times(P<0.001).About aging related gene,the RT-PCR showed that in siRNA group the mRNA expression levels of P16,P21,P53 respectively raised about 1.5(P<0.05)、1.6(P<0.05)、1.6(P<0.001)times,compared to the control group,but expression of P27 mRNA had no obvious change.Conclusion:1.Knockdown Atg7 inhibits the proliferation and differentiation of NSCs.2.Suppressed the autophagy by knocking down Atg7 decreases NSCs vitality suggesting autophagy plays an important role in maintaining the integration of NSCs.