Mechanism Of MiRNA-nov-1/Dhrs3 Mediated MTOR Signaling Pathway Regulating Manganese-induced Apoptosis In N27 Cells

Objective:The rat dopaminergic neuron cell line（"N27"cells for short）was used to construct a stable lentiviral cell line,to explore the regulatory mechanism of mi RNA-nov-1/Dhrs3 in manganese-induced N27 cell apoptosis,as well as the characteristics of key molecular changes in the m TOR signaling pathway,to further elucidate the molecular mechanism of manganese-induced neurotoxicity.Methods:1.N27 cells were exposed to Mn Cl₂ 0μmol/L（blank control group,Control）and 300μmol/L（model group,Model）for 24h.CCK8 was used to detect cell viability,Western blot was used to detect the expression of Dhrs3,apoptosis-related proteins（Caspase-3,Cleaved-caspase-3）and m TOR signaling pathway related proteins（m TOR、p-m TOR、p-S6K1、S6K1、p-4E-BP1、4E-BP1、p-Akt、Akt）.RT-q PCR was used to detect mi RNA-nov-1,Dhrs3 m RNA expression.Nucleic acid sequence alignment software predicts the binding site of mi RNA-nov-1 to Dhrs3.2.N27 cells were divided into the following seven groups:blank control group（Control group）,manganese group（Model group）,manganese+lentivirus empty group（NC group）,manganese+mi RNA-nov-1 overexpression group（mi RNA-nov-1up group）,manganese+mi RNA-nov-1 low expression group（mi RNA-nov-1 sponge group）,manganese+silenced Dhrs3 group（sh-Dhrs3 group）,manganese+mi RNA-nov-1 low expression+silenced Dhrs3 group（mi RNA-nov-1 sponge+sh-Dhrs3 group）.The manganese exposure dose of each group was the best manganese exposure concentration（300μmol/L）,and the exposure time was 24h.and the exposure time was 24h.The cell morphology of each group was observed by an inverted microscope,the apoptosis rate was detected by flow cytometry after Annexin V/PI double staining,the apoptosis rate was detected by Hoechst 33324 fluorescence staining,Western blot was used to detect the expression of Dhrs3,apoptosis-related proteins Caspase-3 and m TOR signaling pathway related proteins（m TOR,p-m TOR,p-S6K1,S6K1,p-4E-BP1,4E-BP1,p-Akt,Akt）,RT-q PCR was used to detect mi RNA-nov-1,Dhrs3,m TOR,S6K1,4E-BP1,Akt m RNA expression.Results:1.The CCK8 experiment found that the effect of manganese on the activity of N27 cells showed a dose-response relationship,and the activity of the cells decreased with the increase of the dose.Western blot results showed that compared with the Control group,Dhrs3 expression in Model group decreased（P<0.05）,apoptosis-related proteins（Caspase-3,Cleaved-caspase-3）and m TOR signaling pathway phosphorylated proteins（p-m TOR,p-S6K1,p-4E-BP1,p-Akt）expression increased（P<0.05）,and total protein（m TOR,S6K1,4E-BP1,Akt）expression remained unchanged（P>0.05）.RT-q PCR results showed that compared with the Control group,the expression of mi RNA-nov-1 in the Model group was up-regulated,and the expression of Dhrs3 was down-regulated,and the difference was statistically significant（P<0.05）.Nucleic acid sequence alignment software predicted the existence of binding sites between mi RNA-nov-1 and Dhrs3.2.Western blot results showed that compared with the Model group and the NC group,The expression of Caspase-3,p-m TOR,p-Akt,p-S6K1,p-4E-BP1 was increased in the mi RNA-nov-1 up group and sh-Dhrs3 group,the expression of Caspase-3 and p-m TOR,p-Akt,p-S6K1,p-4E-BP1 was decreased in the mi RNA-nov-1 sponge group（P<0.05）,the expression of Caspase-3,p-m TOR,p-Akt,p-S6K1,and p-4E-BP1 in the mi RNA-nov-1 sponge+sh-Dhrs3 group was not significant difference（P>0.05）.Compared with the Model group and the NC group,the Dhrs3 expression was decreased in the mi RNA-nov-1 up group and the sh-Dhrs3group,there was no significant difference in Dhrs3 expression in mi RNA-nov-1sponge+sh-Dhrs3 group（P>0.05）.RT-q PCR results showed that compared with the Control group,the expression of mi RNA-nov-1 in the other six groups increased（P<0.05）.Compared with the Model group and the NC group,the mi RNA-nov-1 up group and sh-Dhrs3 the expression of mi RNA-nov-1 was increased（P<0.05）,and there was no significant difference in the expression of mi RNA-nov-1 in the mi RNA-nov-1 sponge+sh-Dhrs3 group（P>0.05）.Compared with the Control group,there was no significant difference in the expression of m TOR,Akt and S6K1 m RNA in Model,NC,mi RNA-nov-1 sponge,sh-Dhrs3 and mi RNA-nov-1 sponge+sh-Dhrs3 groups（P>0.05）.Annexin V/PI double staining flow cytometry results showed that compared with the Control group,the apoptosis rates of the other 6groups were significantly increased（P<0.05）.Compared with the Model group and the NC group,The apoptosis rate of N27 cells was significantly increased in mi RNA-nov-1 up and sh-Dhrs3 group（P<0.05）,the apoptosis rate of N27 cells was significantly decreased in the mi RNA-nov-1 sponge group（P<0.05）,the apoptosis rate of N27 cells was no significant difference in mi RNA-nov-1 sponge+sh-Dhrs3group（P>0.05）.The results of Hoechst 33324 fluorescence staining showed that compared with the Control group,the number of apoptotic cells with dense and dense staining was significantly increased in the other 6 groups.Compared with the Model group and the NC group,The apoptosis rate of N27 cells was significantly increased in mi RNA-nov-1 up and sh-Dhrs3 group,the apoptosis rate of N27 cells was significantly decreased in the mi RNA-nov-1 sponge group,the apoptosis rate of N27cells was no significant difference in mi RNA-nov-1 sponge+sh-Dhrs3 group.Conclusion:1.The expression of mi RNA-nov-1 was up-regulated and the expression of Dhrs3 was down-regulated in the apoptosis of N27 cells induced by manganese;2.mi RNA-nov-1 may promote manganese-induced N27 cells apoptosis by negatively regulating Dhrs3 to activate m TOR signaling pathway.