The Role And Molecular Mechanism Of Pin1 In Neurodegenerative Damage Of Human Glioma Cells Induced By Cobalt Chloride

Objective: To investigate the role of Pin1 in H4 cytotoxicity induced by cobalt chloride,and further elucidate the neurobiological function of Pin1 and its neurodegenerative lesions induced by cobalt chloride by knocking down Pin or overexpressing Pin1 gene.The role and mechanism are better systematic.Methods:(1)H4 cells were respectively treated with 0,100,400,600 ?M Co Cl2 for 24 h and 36 h,CCK8 was used to detect cell proliferation(n=8);Hoechst-33258 staining fluorescence microscopy and flow cytometry(FCM)were used to detect cells apoptosis(n=3);cell cycle was detected by flow cytometry(FCM);Pin1 m RNA expression was detected by q RT-PCR;Pin1,Hif-1a and p-Tau protein expression levels were detected by Western-blot;(2)After infecting H4 cells by knocking down the lentivirus of Pin1,H4 cells successfully knocking down the Pin1 gene were screened.The control group and the experimental group were set to a concentration of 400 ?M Co Cl2.After exposure to knockdown of Pin1 H4 cells for 24 h and 36 h,CCK8 was used to detect cell proliferation(n=6);Hoechst-33258 staining fluorescence microscopy and flow cytometry(FCM)were used to detect cells apoptosis(n=3);cell cycle was detected by flow cytometry(FCM);Pin1 m RNA expression was detected by q RT-PCR;Protein expression levels of Pin1,Hif-1a,Cyclin D1,p-Tau,cis p-Tau and trans p-Tau were detected by western-blot.(3)After infecting H4 cells with a lentivirus overexpressing Pin1,H4 cells that successfully overexpressed the Pin1 gene were screened.The control group and the experimental group were set to a concentration of 400 ?M Co Cl2.After exposure to overexpressing of Pin1 H4 cells for 24 h and 36 h,CCK8 was used to detect cell proliferation(n=6);Hoechst-33258 staining fluorescence microscopy and flow cytometry(FCM)were used to detect cells apoptosis(n=3);cell cycle was detected by flow cytometry(FCM);Pin1 m RNA expression was detected by q RT-PCR;Protein expression levels of Pin1,Hif-1a,Cyclin D1,p-Tau,cis p-Tau and trans p-Tau were detected by western-blot.(4)H4 cells were treated with the inhibitor ATRA or Juglone combined with cobalt chloride.Control group and experimental group were set,Co Cl2 concentration was 200?M,and cell proliferation activity was detected by CCK8 after 72 h exposure to H4 cells(n=6).Hoechst-33258 staining fluorescence microscope and flow cytometry(FCM)were used to detect apoptosis(n=3).Cell cycle was detected by flow cytometry(FCM).The expression of Pin1 m RNA was detected by q RT-PCR.Protein expression levels of Pin1,Hif-1a,cis p-Tau and trans p-Tau were detected by western-blot.Results:(1)Cobalt chloride can significantly inhibit the proliferation of H4 cells,induce apoptosis,cause cell cycle arrest,down-regulate Pin1 m RNA expression,down-regulate Pin1 protein expression,up-regulate Hif-1a protein expression,p-Tau Protein expression was up-regulated,and had a dose-response relationship,and the difference was statistically significant(P<0.05).(2)The results showed that Pin1 gene knockdown effectively increased the ability of Co Cl2 to inhibit the proliferation of H4 cells,increased the ability of Co Cl2 to induce H4 cell death,and promoted H4 cell cycle arrest by Co Cl2.Pin1 gene was compared with the control.Knockdown can promote the decrease of Pin1 m RNA expression in H4 cells induced by Co Cl2.In Co Cl2 treated H4 cells,Pin1 gene knockdown can promote down-regulation of Pin1 protein expression,up-regulation of Hif-1a protein expression,and up-regulation of p-Tau protein expression,and the difference was statistically significant(P<0.05).(3)The results showed that overexpression of Pin1 effectively retarded the ability of Co Cl2 to inhibit the proliferation of H4 cells,retarded the ability of Co Cl2 to induce H4 cell death,and retard the cell cycle arrest induced by Co Cl2.Overexpression of Pin1 gene was compared with the control group.Co Cl2 can be reduced to cause a decrease in the expression of Pin1 m RNA in H4 cells.In the Co Cl2-treated Pin1 gene H4 cells,overexpression of Pin1 gene reduced Pin1 protein down-regulation,Hif-1a protein up-regulation,and p-Tau protein down-regulation,and the difference was statistically significant(P<0.05).(4)The results showed that compared with the Control group,the cells in the Co Cl2 group,ATRA+Co Cl2 group and Juglone+Co Cl2 group showed decreased proliferation activity,increased apoptosis rate and down-regulated Pin1 m RNA expression.In the Co Cl2 group,ATRA group,ATRA+ Co Cl2 group,Juglone group and Juglone+Co Cl2 group,Pin1 protein expression was down-regulated,cis p-tau protein expression was up-regulated,and trans-p-Tau protein expression was down-regulated,which was statistically significant(P<0.05).Hif-1a protein expression was up-regulated in cells from the Co Cl2 group,ATRA+Co Cl2 group,and Juglone+Co Cl2 group compared with the Control group(P<0.05).Compared with Co Cl2 group,the proliferation activity of cells in ATRA+Co Cl2 group and Juglone+Co Cl2 group decreased,apoptosis rate increased,Pin1 m RNA expression down-regulated,Pin1 protein expression down-regulated,hif-1a protein expression down-regulated,and trans-p-Tau protein expression down-regulated.The expression of cis p-tau protein was up-regulated in ATRA+ Co Cl2 group(P<0.05).Compared with ATRA group,ATRA+Co Cl2 group showed decreased cell proliferation,increased apoptosis rate,down-regulated Pin1 m RNA expression,down-regulated Pin1 protein expression,up-regulated hif-1a protein expression,up-regulated cis p-tau protein expression,and down-regulated trans p-tau protein expression,with statistical significance(P <0.05).Compared with the Juglone group,cell proliferation activity decreased,apoptosis rate increased,Pin1 m RNA expression was down-regulated,Pin1 protein expression was down-regulated,hif-1a protein expression was up-regulated,cis p-Tau protein expression was up-regulated,and trans p-Tau protein expression was down-regulated in the Juglone+Co Cl2 group,showing statistical significance(P <0.05).Conclusion:(1)Cobalt chloride can cause degenerative damage to nerve cells and down-regulate the expression of Pin1 m RNA and protein.(2)Knockdown of Pin1 gene can promote the degenerative damage effect of cobalt chloride on nerve cells.(3)Overexpression of Pin1 gene can retard the degenerative damage of cobalt chloride induced nerve cells.(4)Treatment with the Pin1 inhibitor ATRA or Juglone promoted the degenerative damage of cobalt chloride induced nerve cells.(5)Cobalt chloride may down-regulate the function of Pin1 gene through hypoxia,promote the transformation of trans p-Tau protein into cis p-Tau protein,and cause degenerative damage to nerve cells.In conclusion,Pin1 plays a protective role in neurodegenerative damage induced by cobalt chloride,our study provides an important theoretical basis for Pin1 as an important target for the prevention and treatment of neurodegenerative damage caused by cobalt chloride.